Chiral diacylhydrazine ligands for modulating the expression of exogenous genes via an ecdysone receptor complex

ABSTRACT

The present invention provides diacylhydrazine ligands and chiral diacylhydrazine ligands for use with ecdysone receptor-based inducible gene expression systems. Thus, the present invention is useful for applications such as gene therapy, large scale production of proteins and antibodies, cell-based screening assays, functional genomics, proteomics, metabolomics, and regulation of traits in transgenic organisms, where control of gene expression levels is desirable. An advantage of the present invention is that it provides a means to regulate gene expression and to tailor expression levels to suit the user&#39;s requirements.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name“Substitute Sequence Listing.ST25.txt”; Size: 15.0 KB (15,408 bytes);and Date of Creation: Jan. 27, 2009) filed with the application isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the fields of biotechnology, geneticengineering and medicinal chemistry. In one embodiment, this inventionrelates to the field of gene expression. In another embodiment, thisinvention relates to diacylhydrazine ligands and chiral diacylhydrazineligands for natural and mutated nuclear receptors and their use in anuclear receptor-based inducible gene expression system and methods ofmodulating the expression of a gene within a host cell using theseligands and inducible gene expression system.

2. Background

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties. However, the citation ofany reference herein should not be construed as an admission that suchreference is available as “Prior Art” to the present application.

In the field of genetic engineering, precise control of gene expressionis a valuable tool for studying, manipulating, and controllingdevelopment and other physiological processes. Gene expression is acomplex biological process involving a number of specificprotein-protein interactions. In order for gene expression to betriggered, such that it produces the RNA necessary as the first step inprotein synthesis, a transcriptional activator must be brought intoproximity of a promoter that controls gene transcription. Typically, thetranscriptional activator itself is associated with a protein that hasat least one DNA binding domain that binds to DNA binding sites presentin the promoter regions of genes. Thus, for gene expression to occur, aprotein comprising a DNA binding domain and a transactivation domainlocated at an appropriate distance from the DNA binding domain must bebrought into the correct position in the promoter region of the gene.

The traditional transgenic approach utilizes a cell-type specificpromoter to drive the expression of a designed transgene. A DNAconstruct containing the transgene is first incorporated into a hostgenome. When triggered by a transcriptional activator, expression of thetransgene occurs in a given cell type.

Another means to regulate expression of foreign genes in cells isthrough inducible promoters. Examples of the use of such induciblepromoters include the PR1-a promoter, prokaryotic repressor-operatorsystems, immunosuppressive-immunophilin systems, and higher eukaryotictranscription activation systems such as steroid hormone receptorsystems and are described below.

The PR1-a promoter from tobacco is induced during the systemic acquiredresistance response following pathogen attack. The use of PR1-a may belimited because it often responds to endogenous materials and externalfactors such as pathogens, UV-B radiation, and pollutants. Generegulation systems based on promoters induced by heat shock, interferonand heavy metals have been described (Wurn et al., Proc. Natl. Acad.Sci. USA 83:5414-5418 (1986); Arnheiter et al., Cell 62:51-61 (1990);Filmus et al., Nucleic Acids Research 20:27550-27560 (1992)). However,these systems have limitations due to their effect on expression ofnon-target genes. These systems are also leaky.

Prokaryotic repressor-operator systems utilize bacterial repressorproteins and the unique operator DNA sequences to which they bind. Boththe tetracycline (“Tet”) and lactose (“Lac”) repressor-operator systemsfrom the bacterium Escherichia coli have been used in plants and animalsto control gene expression. In the Tet system, tetracycline binds to theTetR repressor protein, resulting in a conformational change thatreleases the repressor protein from the operator which as a resultallows transcription to occur. In the Lac system, a lac operon isactivated in response to the presence of lactose, or synthetic analogssuch as isopropyl-b-D-thiogalactoside. Unfortunately, the use of suchsystems is restricted by unstable chemistry of the ligands, i.e.tetracycline and lactose, their toxicity, their natural presence, or therelatively high levels required for induction or repression. For similarreasons, utility of such systems in animals is limited.

Immunosuppressive molecules such as FK506, rapamycin and cyclosporine Acan bind to immunophilins FKBP12, cyclophilin, etc. Using thisinformation, a general strategy has been devised to bring together anytwo proteins simply by placing FK506 on each of the two proteins or byplacing FK506 on one and cyclosporine A on another one. A synthetichomodimer of FK506 (FK1012) or a compound resulted from fusion ofFK506-cyclosporine (FKCsA) can then be used to induce dimerization ofthese molecules (Spencer et al., Science 262:1019-24 (1993); Belshaw etal., Proc Natl Acad Sci USA 93:4604-7 (1996)). Gal4 DNA binding domainfused to FKBP12 and VP16 activator domain fused to cyclophilin, andFKCsA compound were used to show heterodimerization and activation of areporter gene under the control of a promoter containing Gal4 bindingsites. Unfortunately, this system includes immunosuppressants that canhave unwanted side effects and therefore, limits its use for variousmammalian gene switch applications.

Higher eukaryotic transcription activation systems such as steroidhormone receptor systems have also been employed. Steroid hormonereceptors are members of the nuclear receptor superfamily and are foundin vertebrate and invertebrate cells. Unfortunately, use of steroidalcompounds that activate the receptors for the regulation of geneexpression, particularly in plants and mammals, is limited due to theirinvolvement in many other natural biological pathways in such organisms.In order to overcome such difficulties, an alternative system has beendeveloped using insect ecdysone receptors (EcR).

Growth, molting, and development in insects are regulated by theecdysone steroid hormone (molting hormone) and the juvenile hormones(Dhadialla et al., Annu. Rev. Entomol. 43: 545-569 (1998)). Themolecular target for ecdysone in insects consists of at least ecdysonereceptor (EcR) and ultraspiracle protein (USP). EcR is a member of thenuclear steroid receptor super family that is characterized by signatureDNA and ligand binding domains, and an activation domain (Koelle et al.,Cell, 67:59-77 (1991)). EcR receptors are responsive to a number ofsteroidal compounds such as ponasterone A and muristerone A. Recently,non-steroidal compounds with ecdysteroid agonist activity have beendescribed, including the commercially available insecticidestebufenozide and methoxyfenozide that are marketed world wide by Rohmand Haas Company (see WO 96/27673 and U.S. Pat. No. 5,530,028). Bothanalogs have exceptional safety profiles in other organisms.

The insect ecdysone receptor (EcR) heterodimerizes with Ultraspiracle(USP), the insect homologue of the mammalian retinoid X receptor (RXR),and binds ecdysteroids and ecdysone receptor response elements andactivate transcription of ecdysone responsive genes. The EcR/USP/ligandcomplexes play important roles during insect development andreproduction. The EcR has five modular domains, A/B (transactivation), C(DNA binding, heterodimerization), D (Hinge, heterodimerization), E(ligand binding, heterodimerization and transactivation) and F(transactivation) domains. Some of these domains such as A/B, C and Eretain their function when they are fused to other proteins.

Tightly regulated inducible gene expression systems or “gene switches”are useful for various applications such as gene therapy, large scaleproduction of proteins in cells, cell based high throughput screeningassays, functional genomics and regulation of traits in transgenicplants and animals.

The first version of an EcR-based gene switch used Drosophilamelanogaster EcR (DmEcR) and Mus musculus RXR (MmRXR) and showed thatthese receptors in the presence of steroid, ponasteroneA, transactivatereporter genes in mammalian cell lines and transgenic mice(Christopherson et al., Proc. Natl. Acad. Sci. USA. 89:6314-6318 (1992);No et al., Proc. Natl. Acad. Sci. USA. 93:3346-3351 (1996)). Later, Suhret al., Proc. Natl. Acad. Sci. 95:7999-8004 (1998) showed thatnon-steroidal ecdysone agonist, tebufenozide, induced high level oftransactivation of reporter genes in mammalian cells through Bombyx moriEcR (BmEcR) in the absence of exogenous heterodimer partner.

WO 97/38117 and WO99/58155 disclose methods for modulating theexpression of an exogenous gene in which a DNA construct comprising theexogenous gene and an ecdysone response element is activated by a secondDNA construct comprising an ecdysone receptor that, in the presence of aligand therefore, and optionally in the presence of a receptor capableof acting as a silent partner, binds to the ecdysone response element toinduce gene expression. The ecdysone receptor of choice was isolatedfrom Drosophila melanogaster. Typically, such systems require thepresence of the silent partner, preferably retinoid X receptor (RXR), inorder to provide optimum activation. In mammalian cells, insect ecdysonereceptor (EcR) heterodimerizes with retinoid X receptor (RXR) andregulates expression of target genes in a ligand dependent manner. WO99/02683 discloses that the ecdysone receptor isolated from the silkmoth Bombyx mori is functional in mammalian systems without the need foran exogenous dimer partner.

U.S. Pat. No. 6,265,173 B1 discloses that various members of thesteroid/thyroid superfamily of receptors can combine with Drosophilamelanogaster ultraspiracle receptor (USP) or fragments thereofcomprising at least the dimerization domain of USP for use in a geneexpression system. U.S. Pat. No. 5,880,333 discloses a Drosophilamelanogaster EcR and ultraspiracle (USP) heterodimer system used inplants in which the transactivation domain and the DNA binding domainare positioned on two different hybrid proteins. Unfortunately, theseUSP-based systems are constitutive in animal cells and therefore, arenot effective for regulating reporter gene expression.

In each of these cases, the transactivation domain and the DNA bindingdomain (either as native EcR as in WO 99/02683 or as modified EcR as inWO 97/38117) were incorporated into a single molecule and the otherheterodimeric partners, either USP or RXR, were used in their nativestate.

Drawbacks of the above described EcR-based gene regulation systemsinclude a considerable background activity in the absence of ligands andnon-applicability of these systems for use in both plants and animals(see U.S. Pat. No. 5,880,333). Therefore, a need exists in the art forimproved EcR-based systems to precisely modulate the expression ofexogenous genes in both plants and animals. Such improved systems wouldbe useful for applications such as gene therapy, large-scale productionof proteins and antibodies, cell-based high throughput screening assays,functional genomics and regulation of traits in transgenic animals. Forcertain applications such as gene therapy, it may be desirable to havean inducible gene expression system that responds well to syntheticnon-steroid ligands and at the same time is insensitive to the naturalsteroids. Thus, improved systems that are simple, compact, and dependenton ligands that are relatively inexpensive, readily available, and oflow toxicity to the host would prove useful for regulating biologicalsystems.

Recently, it has been shown that an ecdysone receptor-based induciblegene expression system in which the transactivation and DNA bindingdomains are separated from each other by placing them on two differentproteins results in greatly reduced background activity in the absenceof a ligand and significantly increased activity over background in thepresence of a ligand (see WO 01/70816 A1, incorporated herein in itsentirety by reference). This two-hybrid system is a significantlyimproved inducible gene expression modulation system compared to the twosystems disclosed in applications WO 97/38117 and WO 99/02683. Thetwo-hybrid system exploits the ability of a pair of interacting proteinsto bring the transcription activation domain into a more favorableposition relative to the DNA binding domain such that when the DNAbinding domain binds to the DNA binding site on the gene, thetransactivation domain more effectively activates the promoter (see, forexample, U.S. Pat. No. 5,283,173). Briefly, the two-hybrid geneexpression system comprises two gene expression cassettes; the firstencoding a DNA binding domain fused to a nuclear receptor polypeptide,and the second encoding a transactivation domain fused to a differentnuclear receptor polypeptide. In the presence of ligand, the interactionof the first polypeptide with the second polypeptide effectively tethersthe DNA binding domain to the transactivation domain. Since the DNAbinding and transactivation domains reside on two different molecules,the background activity in the absence of ligand is greatly reduced.

A two-hybrid system also provides improved sensitivity to non-steroidalligands for example, diacylhydrazines, when compared to steroidalligands for example, ponasterone A (“PonA”) or muristerone A (“MurA”).That is, when compared to steroids, the non-steroidal ligands providehigher activity at a lower concentration. In addition, sincetransactivation based on EcR gene switches is often cell-line dependent,it is easier to tailor gene switch systems to obtain maximumtransactivation capability for each application. Furthermore, thetwo-hybrid system avoids some side effects due to overexpression of RXRthat often occur when unmodified RXR is used as a heterodimer receptorpartner. In one two-hybrid system, native DNA binding andtransactivation domains of EcR or RXR are eliminated and as a result,these hybrid molecules have less chance of interacting with othersteroid hormone receptors present in the cell resulting in reduced sideeffects. Additional gene switch systems include those described in thefollowing, each of which is incorporated by reference: U.S. Pat. No.7,091,038; WO2004078924; EP1266015; US20010044151; US20020110861;US20020119521; US20040033600; US20040197861; US20040235097;US20060020146; US20040049437; US20040096942; US20050228016;US20050266457; US20060100416; WO2001/70816; WO2002/29075; WO2002/066612;WO2002/066613; WO2002/066614; WO2002/066615; WO2005/108617; U.S. Pat.No. 6,258,603; US20050209283; US20050228016; US20060020146; EP0965644;U.S. Pat. No. 7,304,162; U.S. Pat. No. 7,304,161; MX234742;KR10-0563143; AU765306; AU2002-248500; and AU2002-306550.

With the improvement in ecdysone receptor-based gene regulation systemsthere is an increase in their use in various applications resulting inincreased demand for ligands with higher activity than those currentlyin existence. U.S. Pat. No. 6,258,603 B1, US 2005/0209283 A1 and US2006/0020146 A1 (and patents cited therein) disclose dibenzoylhydrazineligands. However, a need exists for additional ligands with improvedpharmacological properties. Applicants have discovered chiraldiacylhydrazine ligands which have not previously been described thathave surprising biological activities and the ability to modulate theexpression of transgenes in unexpected ways.

SUMMARY OF THE INVENTION

The present invention provides diacylhydrazine ligands of Formula I andchiral diacylhydrazine ligands of Formula II or III for use withecdysone receptor-based inducible gene expression systems useful formodulating expression of a target gene of interest in a host cell.Chiral diacylhydrazine ligands of the invention are enantiomericallyenriched in either the R- or S-stereoisomer. Applicants have discoveredthese novel chiral diacylhydrazine ligands are surprisingly effective.Thus, the present invention is useful for applications such as genetherapy, large scale production of proteins and antibodies, cell-basedscreening assays, functional genomics, proteomics, metabolomics, andregulation of traits in transgenic organisms, where control of geneexpression levels is desirable. An advantage of the present invention isthat it provides a means to regulate gene expression and to tailorexpression levels to suit the user's requirements.

The present invention pertains to compounds of Formula I

wherein

A is alkoxy, arylalkyloxy or aryloxy;

B is optionally substituted aryl or optionally substituted heteroaryl;and

R¹ and R² are independently optionally substituted alkyl, arylalkyl,hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocycle, optionally substituted aryl or optionallysubstituted heteroaryl;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula II

wherein

A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substitutedaryl or optionally substituted heteroaryl;

B is optionally substituted aryl or optionally substituted heteroaryl;and

R¹ and R² are independently optionally substituted alkyl, arylalkyl,hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocycle, optionally substituted aryl or optionallysubstituted heteroaryl;

with the proviso that R¹ does not equal R²;

wherein the absolute configuration at the asymmetric carbon atom bearingR¹ and R² is predominantly S;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula III

wherein

A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substitutedaryl or optionally substituted heteroaryl;

B is optionally substituted aryl or optionally substituted heteroaryl;and

R¹ and R² are independently optionally substituted alkyl, arylalkyl,hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocycle, optionally substituted aryl or optionallysubstituted heteroaryl;

with the proviso that R¹ does not equal R²;

wherein the absolute configuration at the asymmetric carbon atom bearingR¹ and R² is predominantly R;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In one embodiment, the present invention pertains to(R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide havingan enantiomeric excess of at least 95% or a pharmaceutically acceptablesalt, hydrate, crystalline form or amorphous form thereof.

In another embodiment, the present invention pertains to apharmaceutical composition comprising (R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide havingan enantiomeric excess of at least 95% or a pharmaceutically acceptablesalt, hydrate, crystalline form or amorphous form thereof.

The present invention also pertains to a process for the preparation acompound of Formula IV

wherein

A is arylalkyl, optionally substituted aryl or optionally substitutedheteroaryl;

B is optionally substituted aryl or optionally substituted heteroaryl;

R² is optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted heterocycle,optionally substituted aryl or optionally substituted heteroaryl;

with the proviso that R² does not equal —CR⁸R⁹CHR¹⁰CR¹¹R¹²; and

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently selected from hydrogen,alkyl, cycloalkyl, heterocycle, aryl or heteroaryl;

wherein the asymmetric carbon atom to which R² is attached isenantiomerically enriched in either the R or S isomer;

comprising:

a) reacting a compound of Formula V

with a compound of Formula VIR²HC═N—NH—CO₂R⁷  Formula VI

wherein

X and Y are independently O or NR wherein R is alkyl or aryl;

C_(a) and C_(b) are independently an asymmetric carbon atom of the Sconfiguration or an asymmetric carbon atom of the R configuration;

R¹⁴ and R¹⁵ are independently alkyl or aryl;

R¹³ is halo, hydrogen, alkyl, alkoxy or OSO₂CF₃;

R⁷ is alkyl, arylalkyl or aryl; and

R², R⁸, R⁹, R¹⁰, R¹¹ and R¹² have the same meanings noted above;

to form a compound of Formula VII;

b) reducing the compound of Formula VII to form a compound of FormulaVIII;

c) reacting the compound of Formula VIII with a compound of FormulaB—CO-LG where LG is a leaving group to form a compound of Formula IX;

d) removing the R⁷CO₂— group of the compound of Formula IX to form acompound of Formula X; and

e) reacting the compound of Formula X with a compound of Formula A-CO-LGwhere LG is a leaving group to form a compound of Formula IV.

The present invention also pertains to methods of modulating geneexpression in a host cell using a gene expression modulation system witha diacylhydrazine ligand of Formula I or chiral diacylhydrazine ligandof Formula II or III.

In one embodiment, the present invention relates to the use of adiacylhydrazine ligand of Formula I or chiral diacylhydrazine ligand ofFormula II or III in an inducible gene expression system that has areduced level of background gene expression and responds tosubmicromolar concentrations of the ligand.

In another embodiment, the invention relates to a method of modulatingthe expression of a target gene in a host cell, wherein the host cellcomprises a first gene expression cassette comprising a firstpolynucleotide encoding a first polypeptide comprising:

i) a transactivation domain;

ii) a DNA-binding domain; and

iii) a Group H nuclear receptor ligand binding domain;

and a second gene expression cassette comprising:

i) a response element capable of binding to said DNA binding domain;

ii) a promoter that is activated by the transactivation domain; and

iii) said target gene;

the method comprising contacting said host cell with a diacylhydrazineligand of Formula I or chiral diacylhydrazine ligand of Formula II orIII; wherein the expression of the target gene is modulated.

In another embodiment, the invention relates to a method for regulatingendogenous or heterologous gene expression in a transgenic subjectcomprising contacting a ligand with an ecdysone receptor complex withinthe cells of the subject, wherein the cells further contain a DNAbinding sequence for the ecdysone receptor complex when in combinationwith the ligand and wherein formation of an ecdysone receptorcomplex-ligand-DNA binding sequence complex induces expression of thegene, and where the ligand is a diacylhydrazine ligand of Formula I orchiral diacylhydrazine ligand of Formula II or III; wherein endogenousor heterologous gene expression in a transgenic subject is regulated.

In another embodiment, the invention relates to a method of modulatingthe expression of a target gene in a host cell comprising the steps of:

a) introducing into the host cell a gene expression modulation systemcomprising:

-   -   i) a first gene expression cassette that is capable of being        expressed in a host cell, said first gene expression cassette        comprising a polynucleotide sequence that encodes a first hybrid        polypeptide comprising:

(a) a DNA-binding domain that recognizes a response element associatedwith a gene whose expression is to be modulated; and

(b) an ecdysone receptor ligand binding domain;

-   -   ii) a second gene expression cassette that is capable of being        expressed in the host cell, said second gene expression cassette        comprising a polynucleotide sequence that encodes a second        hybrid polypeptide comprising:

(a) a transactivation domain; and

(b) a chimeric retinoid X receptor ligand binding domain; and

-   -   iii) a third gene expression cassette that is capable of being        expressed in a host cell, said third gene expression cassette        comprising a polynucleotide sequence comprising:

(a) a response element recognized by the DNA-binding domain of the firsthybrid polypeptide;

(b) a promoter that is activated by the transactivation domain of thesecond hybrid polypeptide; and

(c) a gene whose expression is to be modulated; and

b) introducing into the host cell a diacylhydrazine ligand of Formula Ior chiral diacylhydrazine ligand of Formula II or III; wherein theexpression of a gene in a host cell is modulated.

In another embodiment, the present invention relates to a method forproducing a polypeptide comprising the steps of:

a) selecting a cell which is substantially insensitive to exposure to adiacylhydrazine ligand of Formula I or chiral diacylhydrazine ligand ofFormula II or III;

b) introducing into the cell:

-   -   i) a DNA construct comprising:

(a) an exogenous gene encoding the polypeptide; and

(b) a response element;

wherein the gene is under the control of the response element; and

-   -   ii) an ecdysone receptor complex comprising:

(a) a DNA binding domain that binds to the response element;

(b) a binding domain for said ligand; and

(c) a transactivation domain; and

c) exposing the cell to said ligand; wherein a polypeptide is produced.

This embodiment of the invention provides the advantage of temporallycontrolling polypeptide production by the cell. Moreover, in those caseswhen accumulation of such a polypeptide can damage the cell, theexpression of the polypeptide may be limited to short periods byexposing said cell to compounds of the present invention. Such controlis particularly important when the exogenous gene is a therapeutic gene.Therapeutic genes may be called upon to produce polypeptides whichcontrol needed functions, such as the production of insulin in diabeticpatients. They may also be used to produce damaging or even lethalproteins, such as those lethal to cancer cells. Such control may also beimportant when the protein levels produced may constitute a metabolicdrain on growth or reproduction, such as in transgenic plants.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Schematic of switch construct CVBE, and the reporter construct6XEcRE Lac Z. Flanking both constructs are long terminal repeats, G418and puromycin are selectable markers, CMV is the cytomegaloviruspromoter, VBE is coding sequence for amino acids 26-546 from Bombyx moriEcR inserted downstream of the VP16 transactivation domain, 6× EcRE issix copies of the ecdysone response element, lacZ encodes for thereporter enzyme β-galactosidase.

FIGS. 2A-2C: Chiral HPLC comparison of (A) rac-, (B) (R)- and (C)(S)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide.

FIG. 3: Graph showing in vivo comparison of rac-, (R)- and(S)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide on inductionof RheoSwitch® Therapeutic System gene expression in mice. Solid circlesare the S enantiomer, solid triangles are the Racemate, and open circlesare the R enantiomer.

FIG. 4: Table of sample lots or the racemate and R enantiomer of2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide. Samples ofthe R enantiomer obtained by rapid crystallization/precipitation fromeither methanol/water or toluene/heptane yielded the same powder X-raydiffraction pattern (data not shown), and had essentially the samemelting point ([toluene/heptane]166.2-167.1° C., [CH₃OH/H₂O]166.5-167.4°C.) as compared to each other and as compared to a standard obtainedfrom CH₃OH crystallization (165.1-166.5° C.). Samples of two separatepreparations of the racemate obtained by methanol evaporation gave thesame melting point (170-171° C., 169-170° C.) within experimental errorand slight variations in purity.

FIG. 5: Table showing particle size distribution of the micronized Renantiomer of 2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide.

FIG. 6: Table showing particle size distribution of the micronizedracemate of 2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide.

FIG. 7: Table showing bulk and tapped density analysis of micronizedracemate and R enantiomer of 2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide.

FIG. 8: Thermal gravimetric analysis/Differential Thermal Analysis(TGA/DTA) analysis (thermal plot) of micronized racemate and Renantiomer of 2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide demonstratesdifferent crystalline forms. Lot Nos: REH-28-9-1 and REH-28-4-2; sampleweight 7.71994 mg. Signal output in units of uV per mg of sample(uV/mg); thermal gravimetry—percentage weight change of the sample (TG%). Both materials showed an endothermal event on the DTA profile. Theonset temperature for the R enantiomer (163.6° C.) was significantlylower than that of the racemate (171.2° C.). Heat of fusion for the Renantiomer (59.8 uv·s/mg) was also significantly lower than that of theracemate (80.8 uv·s/mg).

FIG. 9: Table showing qualitative comparative solubility test ofracemate and R enantiomer of 2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide inpharmaceutical excipients.

FIG. 10: Table showing results of a thermodynamic equilibrium assay (90°C. for 5 minutes or indicated time; treatment 2) followed by cooling toroom temperature and seeding of racemate and R enantiomer of2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide inpharmaceutical excipients. Treatment 1 is the result of stirring at roomtemperature for ≦2.5 hr.

FIG. 11: Table showing solubility (μM) of racemate and R enantiomer of2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide in 20% PEG1000 in distilled water, pH 7.0.

FIG. 12: Table showing solubility of racemate and R enantiomer of2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide in aqueouspolysorbate 80.

FIG. 13: Table showing bidirectional Permeability of racemate and Renantiomer of 2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide ThroughCaco-2 Cell Monolayers. ^(a)Permeability Classification: (PappA→B)<1.0×10-6 cm/s=LOW; (Papp A→B)>1.0×10-6 cm/s=HIGH; ^(b)SignificantEfflux: Efflux>3.0 and (Papp B→A)>1.0×10-6 cm/s

FIG. 14: Table showing permeability of racemate and R enantiomer of2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide in MDR1-MDCKcells. Classification: A-B Papp>3.0 and Efflux Ratio<3.0: High. A-BPapp>3.0 and 10>Efflux Ratio>3.0: Moderate. A-B Papp>3.0 and EffluxRatio>10: Low A-B Papp<3.0: Low.

FIG. 15: Table showing stability of racemate and R enantiomer of2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide in humanliver microsomes.

FIG. 16: Table showing dosing schedule of racemate and R enantiomer of2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide/Labrasolpreparations in C57BL/6N:Crl mice. ^(a)Dose administration for 9 days(first 18 females/group) or 12 days (second 18 females/group);^(b)Included as possible replacements.

FIG. 17: Micrographs of non-micronized and micronized samples of2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide racemate.Note the 50 micron reference scale.

FIG. 18: Table showing blood serum levels of racemate and R enantiomerof 2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide administeredin Labrasol at 4 dosage levels (3, 10, 30 and 50 mg/kg/day) after 9 daysof drug administration and hours after the daily dose.

FIG. 19: Table showing blood serum levels of racemate and R enantiomerof 2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide administeredin Labrasol at 4 dosage levels (3, 10, 30 and 50 mg/kg/day) after 12days of drug administration and hours after the daily dose.

FIG. 20: Diagrammatic representation of pCMV/GEVY(DEF) plasmid. Theplasmid pCMV/GEVY(DEF) consists of the D, E and F domains fromChoristoneura fumiferana EcR carrying the mutations V390I/Y410E/E274Vfused downstream of the yeast GAL4-DBD (aa 1-147) and placed under thecontrol of the CMV promoter and a downstream SV40 polyadenylation signalin the pBIND vector (Promega Corporation, Madison, Wis., USA). The DEFdomains of EcRs shown were amplified using primers designed based on20-25 nt sequences at the 5′ and 3′ ends. Restriction enzyme sites BamHI and Xba I were added to 5′ and 3′ primers respectively. The PCRproducts were digested with appropriate restriction enzymes and clonedinto pBIND vector

FIG. 21: Diagrammatic representation of pCMV/VP16-Hs-LmRXR vector. Thevector pCMV/VP16-Hs-LmRXR contains a chimeric RXR from Homo sapiens RXRβ(helix 1-8 of E domain) and Locusta migratoria RXR (helix 9-12 of Edomain), fused downstream of VP16-activation domain and placed under thecontrol of the CMV promoter in the pBIND vector.

FIG. 22: Diagrammatic representation of p6xGAL4RE-TTR-SEAP reportervector. The inducible SEAP reporter vector p6xGAL4RE-TTR-SEAP containsthe human secreted alkaline phosphatase reporter gene placed under thecontrol of the inducible promoter consisting of 6 copies of the Gal4response element upstream of the transthyretin (TTR) promoter.

FIG. 23: Diagrammatic representation of Ad-RTS-hIL-12 in which the E1and E3 regions have been deleted and the RTS-IL12 components replace theE1 region

FIG. 24: Graphs showing expression of mIL12p70 in tumor and serum, afterinjection of B16 cells infected with Ad.RheoSP1-mIL12 plasmid, andadministration of R-enantiomer or racemate of diacylhydrazine at dosagesof 0, 3, 10, 30, 50 mg/kg/day. Controls: Untransduced B16-bearing micereceiving ligand only at 50 mg/kg/day for 3 days (Ligand50) anduntreated (no ligand) tumor-bearing mice (TuControl). “L3d then discont.3d”: mice treated with ligand for 3 days then followed for 3 additionaldays during which time ligand is not provided.

FIG. 25: Graphs showing Expression of mIFN-γ in tumor and serum, afterinjection of B16 cells infected with Ad.RheoSP1-mIL12 plasmid, andadministration of R-enantiomer or racemate of diacylhydrazine at dosagesof 0, 3, 10, 30, 50 mg/kg/day. Controls: Untransduced B16-bearing micereceiving ligand only at 50 mg/kg/day for 3 days (Ligand50) anduntreated (no ligand) tumor-bearing mice (TuControl). “L3d then discont.3d”: mice treated with ligand for 3 days then followed for 3 additionaldays during which time ligand is not provided.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to compounds of Formula I

wherein

A is alkoxy, arylalkyloxy or aryloxy;

B is optionally substituted aryl or optionally substituted heteroaryl;and

R¹ and R² are independently optionally substituted alkyl, arylalkyl,hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocycle, optionally substituted aryl or optionallysubstituted heteroaryl;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula II

wherein

A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substitutedaryl or optionally substituted heteroaryl;

B is optionally substituted aryl or optionally substituted heteroaryl;and

R¹ and R² are independently optionally substituted alkyl, arylalkyl,hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocycle, optionally substituted aryl or optionallysubstituted heteroaryl;

with the proviso that R¹ does not equal R²;

wherein the absolute configuration at the asymmetric carbon atom bearingR¹ and R² is predominantly S;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula III

wherein

A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substitutedaryl or optionally substituted heteroaryl;

B is optionally substituted aryl or optionally substituted heteroaryl;and

R¹ and R² are independently optionally substituted alkyl, arylalkyl,hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocycle, optionally substituted aryl or optionallysubstituted heteroaryl;

with the proviso that R¹ does not equal R²;

wherein the absolute configuration at the asymmetric carbon atom bearingR¹ and R² is predominantly R;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula II or III wherein

A is —OC(CH₃)₃, —OCH₂Ph, optionally substituted aryl or optionallysubstituted heteroaryl;

B is optionally substituted aryl;

R¹ is optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₄)alkyl oroptionally substituted (C₂-C₆)alkenyl; and

R² is optionally substituted (C₁-C₆)alkyl, aryl(C₁-C₄)alkyl, optionallysubstituted (C₃-C₇)cycloalkyl or optionally substituted aryl;

with the proviso that R¹ does not equal R²;

wherein

the absolute configuration at the asymmetric carbon atom bearing R¹ andR² is predominantly S in Formula II; and

the absolute configuration at the asymmetric carbon atom bearing R¹ andR² is predominantly R in Formula III;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula II or III wherein

A is optionally substituted aryl or optionally substituted heteroaryl;

B is optionally substituted aryl;

R¹ is optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₄)alkyl oroptionally substituted (C₂-C₆)alkenyl; and

R² is optionally substituted (C₁-C₆)alkyl, aryl(C₁-C₄)alkyl, optionallysubstituted (C₃-C₇)cycloalkyl or optionally substituted aryl;

with the proviso that R¹ does not equal R²;

wherein

the absolute configuration at the asymmetric carbon atom bearing R¹ andR² is predominantly S in Formula II; and

the absolute configuration at the asymmetric carbon atom bearing R¹ andR² is predominantly R in Formula III;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula II or III wherein

A is

R^(3a), R^(3b), R^(3c), R^(3d) and R^(3e) are independently selectedfrom hydrogen, halo, nitro, cyano, hydroxy, amino, optionallysubstituted alkyl, haloalkyl, hydroxyalkyl, arylalkyl, optionallysubstituted cycloalkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocycle, alkoxy, aryloxy,arylalkyloxy, alkylthio, carboxamido, sulfonamido, —COR^(c), —SO₂R^(d),—N(R^(e)COR^(f), —N(R^(e))SO₂R^(g) or —N(R^(e)C═N(R^(h))-amino; or

R^(3a) and R^(3b) taken together with the carbon atoms to which they areattached form a five, six or seven membered fused cycloalkyl orheterocycle ring; or

R^(3b) and R^(3c) taken together with the carbon atoms to which they areattached form a five, six or seven membered fused cycloalkyl orheterocycle ring;

R^(4a), R^(4b), R^(4c), R^(5a), R^(5b), R^(6a), R^(6b) and R^(6c) areindependently selected from hydrogen, halo, nitro, cyano, hydroxy,amino, optionally substituted alkyl, haloalkyl, hydroxyalkyl, arylalkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocycle, alkoxy,aryloxy, arylalkyloxy, alkylthio, carboxamido, sulfonamido, —COR^(c),—SO₂R^(d), —N(R^(e))COR^(f), —N(R^(e))SO₂R^(g) or—N(R^(e))C═N(R^(h))-amino;

X is O or S;

B is

and;

R^(3f), R^(3g), R^(3h), R^(3i) and R^(3j) are independently selectedfrom hydrogen, halo, nitro, cyano, hydroxy, amino, optionallysubstituted alkyl, haloalkyl, hydroxyalkyl, arylalkyl, optionallysubstituted cycloalkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocycle, alkoxy, aryloxy,arylalkyloxy, alkylthio, carboxamido, sulfonamido, —COR^(c), —SO₂R^(d),—N(R^(e))COR^(f), —N(R^(e))SO₂R^(g) or —N(R^(e))C═N(R^(h))-amino; or

R^(3f) and R^(3g) taken together with the carbon atoms to which they areattached form a five, six or seven membered fused cycloalkyl orheterocycle ring; or

R^(3g) and R^(3h) taken together with the carbon atoms to which they areattached form a five, six or seven membered fused cycloalkyl orheterocycle ring;

wherein

R^(c) is hydrogen, hydroxy, haloalkyl, hydroxyalkyl, arylalkyl,optionally substituted alkyl, optionally substituted cycloalkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted heterocycle, optionally substituted aryl,optionally substituted heteroaryl, alkoxy, aryloxy or arylalkyloxy;

R^(d) is haloalkyl, hydroxyalkyl, arylalkyl, optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedheterocycle, optionally substituted aryl or optionally substitutedheteroaryl;

R^(e) is hydrogen, haloalkyl, hydroxyalkyl, arylalkyl, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocycle, optionally substituted aryl or optionallysubstituted heteroaryl;

R^(f) is hydrogen, haloalkyl, hydroxyalkyl, arylalkyl, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocycle, optionally substituted aryl, optionallysubstituted heteroaryl, alkoxy, aryloxy, arylalkyloxy or amino;

R^(g) is haloalkyl, hydroxyalkyl, arylalkyl, optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedheterocycle, optionally substituted aryl, optionally substitutedheteroaryl or amino;

R^(h) is hydrogen, alkyl, aryl, cyano or nitro;

R¹ is optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₄)alkyl oroptionally substituted (C₂-C₆)alkenyl; and

R² is optionally substituted (C₁-C₆)alkyl, aryl(C₁-C₄)alkyl, optionallysubstituted (C₃-C₇)cycloalkyl or optionally substituted aryl;

with the proviso that R¹ does not equal R²;

wherein

the absolute configuration at the asymmetric carbon atom bearing R¹ andR² is predominantly S in Formula II; and

the absolute configuration at the asymmetric carbon atom bearing R¹ andR² is predominantly R in Formula III;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula II wherein

A, B, R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f), R^(3g), R^(3h),R^(3i), R^(3j), R^(4a), R^(4b), R^(4c), R^(5a), R^(5b), R^(6a), R^(6b),R^(6c) and X have the same meanings as noted above;

R¹ is (C₁-C₆)alkyl, hydroxy(C₁-C₄)alkyl or (C₂-C₄)alkenyl; and

R² is optionally substituted (C₃-C₇)cycloalkyl;

wherein the absolute configuration at the asymmetric carbon atom bearingR¹ and R² is predominantly S;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula III wherein

A, B, R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f), R^(3g), R^(3h),R^(3i), R^(3j), R^(4a), R^(4b), R^(4c), R^(5a), R^(5b), R^(6a), R^(6b),R^(6c) and X have the same meanings as noted above;

R¹ is (C₁-C₆)alkyl, hydroxy(C₁-C₄)alkyl or (C₂-C₄)alkenyl; and

R² is optionally substituted (C₁-C₆)alkyl;

with the proviso that R¹ does not equal R²;

wherein the absolute configuration at the asymmetric carbon atom bearingR¹ and R² is predominantly R;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In one embodiment, the present invention pertains to enantiomericallyenriched compounds of Formula II wherein

A is

B is

R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f), R^(3g), R^(3h), R^(3i)and R^(3j) are independently selected from hydrogen, halo, (C₁-C₄)alkylor (C₁-C₄)alkoxy;

R¹ is (C₁-C₆)alkyl, hydroxy(C₁-C₄)alkyl or (C₂-C₄)alkenyl; and

R² is optionally substituted (C₃-C₇)cycloalkyl

wherein the absolute configuration at the asymmetric carbon atom bearingR¹ and R² is predominantly S;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula III wherein

A is

B is

R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f), R^(3g), R^(3h), R^(3i)and R^(3j) are independently selected from hydrogen, halo, (C₁-C₄)alkylor (C₁-C₄)alkoxy;

R¹ is (C₁-C₆)alkyl, hydroxy(C₁-C₄)alkyl or (C₂-C₄)alkenyl; and

R² is optionally substituted (C₁-C₆)alkyl;

wherein the absolute configuration at the asymmetric carbon atom bearingR¹ and R² is predominantly R;

or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula II having an enantiomericexcess of at least 50%, 75%, 85%, 95% or >99% of the S-isomer. In oneembodiment, the compound of Formula II consists essentially of theS-isomer.

In another embodiment, the present invention pertains toenantiomerically enriched compounds of Formula III having anenantiomeric excess of at least 50%, 75%, 85%, 95% or >99% of theR-isomer. In one embodiment, the compound of Formula III consistsessentially of the R-isomer.

In another embodiment, the present invention pertains to apharmaceutical composition comprising the compound of Formula I, II orIII.

In one embodiment, the present invention pertains to(R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide havingan enantiomeric excess of at least 95% or of at least 99%, or apharmaceutically acceptable salt, hydrate, crystalline form or amorphousform thereof. In one embodiment, the compound is enantiomerically pure.

In another embodiment, the present invention pertains to apharmaceutical composition comprising (R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide havingan enantiomeric excess of at least 95% or of at least 99%, or apharmaceutically acceptable salt, hydrate, crystalline form or amorphousform thereof. In one embodiment, the compound is enantiomerically pure.

It has been unexpectedly discovered that enantiomerically pure(R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide has acombination of beneficial properties which make it uniquely suitable forin vivo pharmaceutical use. Such properties include a lower meltingpoint and lower heat of fusion compared to the racemate (Example 68);better solubility in many liquid pharmaceutical excipients compared tothe racemate (Example 68); better permeability in certain cells comparedto the racemate (Example 69); is more likely to be able to cross theblood-brain barrier than the racemate (Example 69); better stability tohepatocyte metabolism compared to the racemate (Example 70); whenadministered orally as a Labrasol solution or suspension, achievessignificantly higher plasma levels compared to the racemate (Example71); and achieves much greater in vivo gene expression by activation ofa EcR-based gene switch compared to the racemate (Example 72).

The present invention also pertains to a process for the preparation acompound of Formula IV

wherein

A is arylalkyl, optionally substituted aryl or optionally substitutedheteroaryl;

B is optionally substituted aryl or optionally substituted heteroaryl;

R² is optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted heterocycle,optionally substituted aryl or optionally substituted heteroaryl;

with the proviso that R² does not equal —CR⁸R⁹CHR¹⁰CR¹¹R¹²; and

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently selected from hydrogen,alkyl, cycloalkyl, heterocycle, aryl or heteroaryl;

wherein the asymmetric carbon atom to which R² is attached isenantiomerically enriched in either the R or S isomer;

comprising:

a) reacting a compound of Formula V

with a compound of Formula VIR²HC═N—NH—CO₂R⁷  Formula VI

wherein

X and Y are independently O or NR wherein R is alkyl or aryl;

C_(a) and C_(b) are independently an asymmetric carbon atom of the Sconfiguration or a asymmetric carbon atom of the R configuration;

R¹⁴ and R¹⁵ are independently alkyl or aryl;

R¹³ is halo, hydrogen, alkyl, alkoxy or OSO₂CF₃;

R⁷ is alkyl, arylalkyl or aryl; and

R², R⁸, R⁹, R¹⁰, R¹¹ and R¹² have the same meanings noted above;

to form a compound of Formula VII;

b) reducing the compound of Formula VII to form a compound of FormulaVIII;

c) reacting the compound of Formula VIII with a compound of FormulaB—CO-LG where LG is a leaving group to form a compound of Formula IX;

d) removing the R⁷CO₂— group of the compound of Formula IX to form acompound of Formula X; and

e) reacting the compound of Formula X with a compound of Formula A-CO-LGwhere LG is a leaving group, to form a compound of Formula IV.

In another embodiment, the present invention pertains to a process forthe preparation a compound of Formula IV wherein R⁸, R⁹, R¹⁰, R¹¹ andR¹² are hydrogen.

In another embodiment, the present invention pertains to a process forthe preparation a compound of Formula IV wherein

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are hydrogen;

X is NR and R is methyl;

Y is O;

R¹⁴ is methyl;

R¹⁵ is phenyl; and

each of C_(a) and C_(b) is of the R configuration.

In another embodiment, the present invention pertains to a process forthe preparation a compound of Formula IV wherein

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are hydrogen;

X is NR and R is methyl;

Y is O;

R¹⁴ is methyl;

R¹⁵ is phenyl; and

each of C_(a) and C_(b) is of the S configuration.

In another embodiment, the present invention pertains to a process forthe preparation a compound of Formula IV wherein

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are hydrogen;

X is NR and R is methyl;

Y is O;

R¹⁴ is methyl;

R¹⁵ is phenyl;

each of C_(a) and C_(b) is of the S configuration; and

B is 3,5-di-methylphenyl.

In another embodiment, the present invention pertains to a process forthe preparation a compound of Formula IV wherein

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are hydrogen;

X is NR and R is methyl;

Y is O;

R¹⁴ is methyl;

R¹⁵ is phenyl;

each of C_(a) and C_(b) is of the S configuration; and

A is 2-ethyl-3-methoxyphenyl.

In another embodiment, the present invention pertains to a process forthe preparation a compound of Formula IV wherein

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are hydrogen;

X is NR and R is methyl;

Y is O;

R¹⁴ is methyl;

R¹⁵ is phenyl;

each of C_(a) and C_(b) is of the S configuration; and

R² is tert-butyl.

The present invention also pertains to a process for the preparation ofa compound of Formula II or III comprising:

a) reacting an acyl hydrazine of formula XI

with a ketone of Formula XII

to form a compound of Formula XIII;

wherein R¹ does not equal R²;

b) reducing the compound of Formula XIII in the presence of a chiralcatalyst to form a compound of Formula S-XIV or R-XIV; and

c) reacting the compound of Formula S-XIV or R-XIV with a compound ofFormula B—CO-LG where LG is a leaving group to form a compound ofFormula II or III.

Diacylhydrazines of Formula I of the invention include, but are notlimited to:

-   rac-N′-(1-tert-Butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic    acid benzyl ester; or    rac-N′-(1-tert-Butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic    acid tert-butyl ester.

Chiral diacylhydrazines of Formula II or III of the present inventioninclude, but are not limited to:

-   (S)-3,5-Dimethyl-benzoic acid    N-(1-cyclohexyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;-   (S)-3,5-Dimethyl-benzoic acid    N-(1-cyclohexyl-butyl)-N′-(3-methoxy-benzoyl)-hydrazide;-   (S)-3,5-Dimethyl-benzoic acid    N-(1-cyclohexyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide.-   (R)—N′-(1-tert-Butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic    acid benzyl ester;-   (R)—N′-(1-tert-Butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic    acid tert-butyl ester;-   (R)—N′-(1-tert-Butyl-4-hydroxy-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic    acid benzyl ester;-   (R)—N′-(1-tert-Butyl-4-hydroxy-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazine    carboxylic acid benzyl ester;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-methoxy-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-chloro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N′-(2-bromo-benzoyl)-N-(1-tert-butyl-butyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methoxy-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-chloro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-ethyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-methoxy-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-chloro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2,6-difluoro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3,4-dimethoxy-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3,5-difluoro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-methyl-benzo[1,3]dioxole-5-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(naphthalene-1-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(naphthalene-2-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(thiophene-2-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2,5-dimethyl-furan-3-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-chloro-pyridine-3-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(6-chloro-pyridine-3-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethoxy-4-methyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;    and-   (R)-3,5-Dimethyl-benzoic acid    N′-(4-ethyl-benzoyl)-N-(1-phenethyl-but-3-enyl)-hydrazide.

Chiral diacylhydrazines of Formula II and III may contain chiralitycenters in other portions of the molecule (i.e., not at the asymmetriccarbon atom bearing R¹ and R²). All possible enantiomers, diastereomersand stereoisomers are intended to be encompassed within the scope of thepresent invention.

The present invention also pertains to methods of modulating geneexpression in a host cell using a gene expression modulation system witha diacylhydrazine ligand of Formula I or chiral diacylhydrazine ligandof Formula II or III.

In one embodiment, the present invention relates to the use of adiacylhydrazine ligand of Formula I or chiral diacylhydrazine ligand ofFormula II or III in an inducible gene expression system that has areduced level of background gene expression in the absence of ligand andresponds to submicromolar concentrations of the ligand.

In another embodiment, the present invention relates to a method ofmodulating the expression of a target gene in a host cell, wherein thehost cell comprises a first gene expression cassette comprising a firstpolynucleotide encoding a first polypeptide comprising:

i) a transactivation domain;

ii) a DNA-binding domain; and

iii) a Group H nuclear receptor ligand binding domain;

and a second gene expression cassette comprising:

i) a response element capable of binding to said DNA binding domain;

ii) a promoter that is activated by the transactivation domain; and

iii) said target gene;

the method comprising contacting said host cell with a diacylhydrazineligand of Formula I or chiral diacylhydrazine ligand of Formula II orIII; wherein the expression of the target gene is modulated.

In another embodiment, the present invention relates to a method ofmodulating the expression of a gene of interest in a host cell, whereinthe host cell comprises a first recombinant gene expression cassettecomprising a first polynucleotide encoding a first polypeptidecomprising:

i) a DNA-binding domain; and

ii) a Group H nuclear receptor ligand binding domain;

and a second recombinant gene expression cassette comprising:

i) a response element capable of binding to said DNA binding domain;

ii) a promoter; and

iii) said gene of interest;

the method comprising contacting said host cell with a diacylhydrazineligand of Formula I or chiral diacylhydrazine ligand of Formula II orIII; wherein the expression of the gene of interest is modulated.

In another embodiment, the invention relates to a method for regulatingendogenous or heterologous gene expression in a transgenic subjectcomprising contacting a ligand with an ecdysone receptor complex withinthe cells of the subject, wherein the cells further contain a DNAbinding sequence for the ecdysone receptor complex when in combinationwith the ligand and wherein formation of an ecdysone receptorcomplex-ligand-DNA binding sequence complex induces expression of thegene, and where the ligand is a diacylhydrazine ligand of Formula I orchiral diacylhydrazine ligand of Formula II or III, wherein endogenousor heterologous gene expression in a transgenic subject is regulated.

In one embodiment, the cells are autologous cells, i.e. the cells areobtained from the subject and transfected with the ecdysone receptorcomplex and DNA binding sequence. The transfected autologous cells arethen implanted back into the subject which is then treated with theligand. The autologous cells may be implanted in any of a number ofways, including by infusion or injection, e.g. direct injection. In oneembodiment, the autologous cells are implanted by intra-tumoralinjection.

In another embodiment, the invention relates to a method for regulatingendogenous or heterologous gene expression in a transgenic subjectcomprising contacting a ligand with a chimeric ecdysone receptor complexwithin the cells of the subject, wherein the cells further contain a DNAbinding sequence further comprising a promoter for the ecdysone receptorcomplex when in combination with the ligand and wherein formation of anecdysone receptor complex-ligand-DNA binding sequence complex inducesexpression of the gene, and where the ligand is a diacylhydrazine ligandof Formula I or chiral diacylhydrazine ligand of Formula II or III,wherein endogenous or heterologous gene expression in a transgenicsubject is regulated.

In another embodiment the transgenic subject is a plant, insect, animalor mammal, e.g human or veterinary animal such as a cow, pig, sheep,goat, horse, dog or cat.

In another embodiment, the invention relates to a method of regulatingtransgene expression in a transgenic subject, wherein said transgenicsubject comprises a recombinant ecdysone receptor complex capable ofregulating transgene expression; the method comprising administering tosaid subject an effective amount of a diacylhydrazine ligand of FormulaI or chiral diacylhydrazine ligand of Formula II or III.

In another embodiment, the invention relates to a method of modulatingthe expression of a target gene in a host cell comprising the steps of:

a) introducing into the host cell a gene expression modulation systemcomprising:

-   -   i) a first gene expression cassette that is capable of being        expressed in a host cell, said first gene expression cassette        comprising a polynucleotide sequence that encodes a first hybrid        polypeptide comprising:

(a) a DNA-binding domain that recognizes a response element associatedwith a gene whose expression is to be modulated; and

(b) an ecdysone receptor ligand binding domain;

-   -   ii) a second gene expression cassette that is capable of being        expressed in the host cell, said second gene expression cassette        comprising a polynucleotide sequence that encodes a second        hybrid polypeptide comprising:

(a) a transactivation domain; and

(b) a chimeric retinoid X receptor ligand binding domain; and

-   -   iii) a third gene expression cassette that is capable of being        expressed in a host cell, said third gene expression cassette        comprising a polynucleotide sequence comprising:

(a) a response element recognized by the DNA-binding domain of the firsthybrid polypeptide;

(b) a promoter that is activated by the transactivation domain of thesecond hybrid polypeptide; and

(c) a gene whose expression is to be modulated; and

b) introducing into the host cell a diacylhydrazine ligand of Formula Ior chiral diacylhydrazine ligand of Formula II or III; wherein theexpression of a gene in a host cell is modulated.

In another embodiment, the present invention relates to a method forproducing a polypeptide comprising the steps of:

a) selecting a host cell which is substantially insensitive to exposureto a diacylhydrazine ligand of Formula I or chiral diacylhydrazineligand of Formula II or III;

b) introducing into the host cell:

-   -   i) a DNA construct comprising:

(a) an exogenous gene encoding the polypeptide; and

(b) a response element;

wherein the gene is under the control of the response element; and

-   -   ii) an ecdysone receptor complex comprising:

a) a DNA binding domain that binds to the response element;

b) a binding domain for said compound; and

c) a transactivation domain; and

c) exposing the cell to said compound; wherein a polypeptide isproduced.

In another embodiment, the invention pertains to a method wherein theecdysone receptor complex or ecdysone receptor ligand binding domaincomprises a mutation.

The abbreviations used herein have their conventional meaning within thechemical and biological arts, unless otherwise specified. For example:“h” means hour(s), “min” means minute(s), “sec” means second(s), “d”means day(s), “μL” means microliter(s), “mL” means milliliter(s), “L”means liter(s), “μM” means micromolar, “mM” means millimolar, “M” meansmolar, “mol” means moles, “mmol” means millimoles, “μg” meansmicrogram(s), “mg” means milligram(s), “A” means adenine or adenosine,“T” means thymine or thymidine, “G” means guanine or guanosine, “C”means cytidine or cytosine, “×g” means times gravity, “nt” meansnucleotide(s), “aa” means amino acid(s), “bp” means base pair(s), “kb”means kilobase(s), “k” means kilo, “μ” means micro, “° C.” means degreesCelsius, “THF” means tetrahydrofuran, “DME” means dimethoxyethane, “DMF”means dimethylformamide, “NMR” means nuclear magnetic resonance, “psi”refers to pounds per square inch, and “TLC” means thin layerchromatography.

The term “alkyl” as used herein by itself or part of another grouprefers to a straight-chain or branched saturated aliphatic hydrocarbonhaving from one to ten carbons or the number of carbons designated(C₁-C₁₀ means 1 to 10 carbons). Exemplary alkyl groups include methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, n-hexyl, isohexyl, n-heptyl, 4,4-dimethylpentyl, n-octyl,2,2,4-trimethylpentyl, nonyl, decyl and the like.

The term “optionally substituted alkyl” as used herein by itself or partof another group refers to an alkyl as defined above that is optionallysubstituted with one to three substituents independently selected fromnitro, cyano, amino, optionally substituted cycloalkyl, optionallysubstituted heteroaryl, optionally substituted heterocycle, alkoxy,aryloxy, arylalkyloxy, alkylthio, carboxamido, sulfonamido, —COR^(c),—SO₂R^(d), —N(R^(e))COR^(f), —N(R^(e))SO₂R^(g) or—N(R^(e))C═N(R^(h))-amino. Exemplary substituted alkyl groups include—CH₂OCH₃, —CH₂CH₂NH₂, —CH₂CH₂CN, —CH₂SO₂CH₃ and the like.

The term “haloalkyl” as used herein by itself or part of another grouprefers to an alkyl as defined above having one to six halosubstitutents. Exemplary haloalkyl groups include, trifluoromethyl,—CH₂CH₂F and the like.

The term “hydroxyalkyl” as used herein by itself or part of anothergroup refers to an alkyl as defined above having one to three hydroxysubstitutents, typically one hydroxy substituent. Exemplary hydroxyalkylgroups include hydroxymethyl, hydroxyethyl, hydroxypropyl, and the like.

The term “arylalkyl” as used herein by itself or part of another grouprefers to an alkyl as defined above having one to three arylsubstitutents (said aryl substituents may be optionally substituted asdescribed below), typically one or two aryl substituents. Exemplaryarylalkyl groups include, for example, benzyl, phenylethyl,4-fluorophenylethyl, phenylpropyl, diphenylmethyl and the like.

The term “cycloalkyl” as used herein by itself or part of another grouprefers to saturated and partially unsaturated (containing one or twodouble bonds) cyclic hydrocarbon groups containing one to three ringshaving from three to twelve carbon atoms or the number of carbonsdesignated. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin,adamantyl and the like.

The term “optionally substituted cycloalkyl” as used herein by itself orpart of another group refers to a cycloalkyl as defined above that isoptionally substituted with one to three substituents independentlyselected from halo, nitro, cyano, hydroxy, amino, optionally substitutedalkyl, haloalkyl, hydroxyalkyl, arylalkyl, optionally substitutedcycloalkyl, optionally substituted alkenyl, optionally substitutedalkynyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocycle, alkoxy, aryloxy, arylalkyloxy,alkylthio, carboxamido, sulfonamido, —COR^(c), —SO₂R^(d),—N(R^(e))COR^(f), —N(R^(e))SO₂R^(g) or —N(R^(e))C═N(R^(h))-amino.Exemplary optionally substituted cycloalkyl groups include

and the like. An optionally substituted cycloalkyl may be fused to anaryl group to provide an optionally substituted aryl as described below.

The term “alkenyl” as used herein by itself or part of another grouprefers to an alkyl group as defined above containing one to threecarbon-to-carbon double bonds, typically one or two double bonds.Exemplary alkenyl groups include —CH═CH₂, —CH₂CH═CH₂, —CH₂CH₂CH═CH₂,—CH₂CH₂CH═CHCH₃ and the like.

The term “optionally substituted alkenyl” as used herein by itself orpart of another group refers to an alkenyl as defined above that isoptionally substituted with one to three substituents independentlyselected from halo, nitro, cyano, hydroxy, amino, optionally substitutedalkyl, haloalkyl, hydroxyalkyl, arylalkyl, optionally substitutedcycloalkyl, optionally substituted alkenyl, optionally substitutedalkynyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocycle, alkoxy, aryloxy, arylalkyloxy,alkylthio, carboxamido, sulfonamido, —COR^(c), —SO₂R^(d),—N(R^(e))COR^(f), —N(R^(e))SO₂R^(g) or —N(R^(e))C═N(R^(h))-amino.Exemplary optionally substituted alkenyl groups include —CH═CHPh,—CH₂CH═CHPh and the like.

The term “alkynyl” as used herein by itself or part of another grouprefers to an alkyl group as defined above containing one to threecarbon-to-carbon triple bonds, typically one or two triple bonds.Exemplary alkynyl groups include —C≡CH, —C≡CCH₃, —CH₂C≡CH, —CH₂CH₂C≡CHand —CH₂CH₂C≡CCH₃.

The term “optionally substituted alkynyl” as used herein by itself orpart of another group refers to an alkynyl as defined above that isoptionally substituted with one to three substituents independentlyselected from halo, nitro, cyano, hydroxy, amino, optionally substitutedalkyl, haloalkyl, hydroxyalkyl, arylalkyl, optionally substitutedcycloalkyl, optionally substituted alkenyl, optionally substitutedalkynyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocycle, alkoxy, aryloxy, arylalkyloxy,alkylthio, carboxamido, sulfonamido, —COR^(c), —SO₂R^(d),—N(R^(e))COR^(f), —N(R^(e))SO₂R^(g) or —N(R^(e))C═N(R^(h))-amino.Exemplary optionally substituted alkenyl groups include —C≡CPh,—CH₂C≡CPh and the like.

The term “aryl” as used herein by itself or part of another group refersto monocyclic and bicyclic aromatic ring systems having from six totwelve carbon atoms such as phenyl (abbreviated as Ph), 1-naphthyl and2-naphthyl.

The term “optionally substituted aryl” as used herein by itself or partof another group refers to an aryl as defined above that is optionallysubstituted with one to five substituents, typically one to threesubstitutents, independently selected from halo, nitro, cyano, hydroxy,amino, optionally substituted alkyl, haloalkyl, hydroxyalkyl, arylalkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocycle, alkoxy,aryloxy, arylalkyloxy, alkylthio, carboxamido, sulfonamido, —COR^(c),—SO₂R^(d), —N(R^(e)COR^(f), —N(R^(e))SO₂R^(g) or—N(R^(e)C═N(R^(h))-amino. Exemplary optionally substituted aryl groupsinclude 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl,2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-chlorophenyl,4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl, 4-chlorophenyl,2,6-di-fluorophenyl, 2,6-di-chlorophenyl, 2-methyl, 3-methoxyphenyl,2-ethyl, 3-methoxyphenyl, 3,4-di-methoxyphenyl, 3,5-di-fluorophenyl3,5-di-methylphenyl and 3,5-dimethoxy, 4-methylphenyl and the like. Theterm optionally substituted aryl is meant to include groups having fusedoptionally substituted cycloalkyl and fused optionally substitutedheterocycle rings. Examples include

and the like.

The term “heteroaryl” as used herein by itself or part of another grouprefers to monocyclic and bicyclic aromatic ring systems having from fiveto fourteen carbon atoms and one, two, three or four heteroatomsindependently selected from oxygen, nitrogen and sulfur. Exemplaryheteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 4-benzimidazolyl,5-benzimidazolyl, 2-benzthiazolyl, 4-benzthiazolyl, 5-benzthiazolyl,5-indolyl, 5-indolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 2-quinolyl3-quinolyl, 6-quinolyl and the like. The term heteroaryl is meant toinclude possible N-oxides. Exemplary N-oxides include pyridyl N-oxideand the like.

The term “optionally substituted heteroaryl” as used herein by itself orpart of another group refers to a heteroaryl as defined above that isoptionally substituted at any available carbon atom with one to foursubstituents, typically one or two substituents, independently selectedfrom halo, nitro, cyano, hydroxy, amino, optionally substituted alkyl,haloalkyl, hydroxyalkyl, arylalkyl, optionally substituted cycloalkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocycle, alkoxy, aryloxy, arylalkyloxy,alkylthio, carboxamido, sulfonamido, —COR^(c), —SO₂R^(d),—N(R^(e))COR^(f), —N(R^(e))SO₂R^(g) or —N(R^(e)C═N(R^(h))-amino.Exemplary substituted heteroaryl groups include

and the like.

The specific chemical nature of the optionally substituted aryl andoptionally substituted heteroaryl groups for the terminal moieties A andB in the prior identified chiral diacylhydrazine ligands is not narrowlycritical, and as noted, a wide variety of substituents are contemplated.Preferably, substituents for the optionally substituted aryl andoptionally substituted heteroaryl are selected such that the totalnumber of carbon and heteroatoms is no more than about twenty, morepreferably no more than about fifteen.

The term “heterocycle” as used herein by itself or part of another grouprefers to saturated and partially unsaturated (containing one or twodouble bonds) cyclic groups containing one to three rings having fromtwo to twelve carbon atoms and one or two oxygen, sulfur or nitrogenatoms. The heterocycle can be optionally linked to the rest of themolecule through a carbon atom or a nitrogen atom. Exemplary heterocyclegroups include

and the like.

The term “optionally substituted heterocycle” as used herein by itselfor part of another group refers to a heterocycle as defined above thatis optionally substituted with one to four substituents, typically oneor two substituents independently selected from halo, nitro, cyano,hydroxy, amino, optionally substituted alkyl, haloalkyl, hydroxyalkyl,arylalkyl, optionally substituted cycloalkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocycle,alkoxy, aryloxy, arylalkyloxy, alkylthio, carboxamido, sulfonamido,—COR^(c), —SO₂R^(d), —N(R^(e))COR^(f), —N(R^(e))SO₂R^(g) or—N(R^(e))C═N(R^(h))-amino. Exemplary optionally substituted heterocyclegroups include

and the like. An optionally substituted heterocycle may be fused to anaryl group to provide an optionally substituted aryl as described above.

The term “alkoxy” as used herein by itself or part of another grouprefers to a hydroxyalkyl, haloalkyl, arylalkyl, optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted alkenylor optionally substituted alkynyl attached to a terminal oxygen atom.Exemplary alkoxy groups include methoxy, tert-butoxy, —OCH₂CH═CH₂ andthe like.

The term “aryloxy” as used herein by itself or part of another grouprefers to an optionally substituted aryl attached to a terminal oxygenatom. Exemplary aryloxy groups include phenoxy and the like.

The term “arylalkyloxy” as used herein by itself or part of anothergroup refers to an arylalkyl attached to a terminal oxygen atom.Exemplary arylalkyloxy groups include benzyloxy and the like.

The term “alkylthio” as used herein by itself or part of another grouprefers to a hydroxyalkyl, haloalkyl, arylalkyl, optionally substitutedalkyl, optionally substituted alkenyl or optionally substituted alkynylattached to a terminal sulfur atom. Exemplary alkyl groups include —SCH₃and the like.

The term “halo” or “halogen” as used herein by itself or part of anothergroup refers to fluoro, chloro, bromo or iodo.

The term “amino” as used herein by itself or part of another grouprefers to a radical of formula —NR^(a)R^(b) wherein R^(a) and R^(b) areindependently hydrogen, haloalkyl, hydroxyalkyl, arylalkyl, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted heterocycle, optionally substituted aryl or optionallysubstituted heteroaryl; or R^(a) and R^(b) taken together with thenitrogen atom to which they are attached form a four to seven memberedheterocycle. Exemplary amino groups include —NH₂, —N(H)CH₃, —N(CH₃)₂,—N(H)CH₂Ph and the like.

The term “carboxamido” as used herein by itself or part of another grouprefers to a radical of formula —CO-amino. Exemplary carboxamido groupsinclude —CONH₂, —CON(H)CH₃, —CON(H)Ph and the like.

The term “sulfonamido” as used herein by itself or part of another grouprefers to a radical of formula —SO₂-amino. Exemplary sulfonamido groupsinclude —SO₂NH₂, —SO₂N(H)CH₃, —SO₂N(H)Ph and the like.

In reference to the optionally substituted groups described above, R^(c)is hydrogen, hydroxy, haloalkyl, hydroxyalkyl, arylalkyl, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocycle, optionally substituted aryl, optionallysubstituted heteroaryl, alkoxy, aryloxy or arylalkyloxy; R^(d) ishaloalkyl, hydroxyalkyl, arylalkyl, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted heterocycle,optionally substituted aryl or optionally substituted heteroaryl; R^(e)is hydrogen, haloalkyl, hydroxyalkyl, arylalkyl, optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedheterocycle, optionally substituted aryl or optionally substitutedheteroaryl; R^(f) is hydrogen, haloalkyl, hydroxyalkyl, arylalkyl,optionally substituted alkyl, optionally substituted cycloalkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted heterocycle, optionally substituted aryl,optionally substituted heteroaryl, alkoxy, aryloxy, arylalkyloxy oramino; R^(g) is haloalkyl, hydroxyalkyl, arylalkyl, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocycle, optionally substituted aryl, optionallysubstituted heteroaryl or amino; and R^(h) is hydrogen, alkyl, aryl,cyano or nitro.

Throughout the specification, groups and optional substituents thereofare chosen to provide stable moieties and compounds.

The compounds of the present invention may contain unnatural proportionsof atomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are intended to beencompassed within the scope of the present invention.

The compounds of the present invention may form salts which are alsowithin the scope of this invention. Reference to a compound of thepresent invention herein is understood to include reference to saltsthereof, unless otherwise indicated. The term “salt(s)” as used hereindenotes acidic and/or basic salts formed with inorganic and/or organicacids and bases. In addition, when a compound of Formula I, II or IIIcontains both a basic moiety and an acidic moiety, zwitterions (“innersalts”) may be formed and are included within the term “salt(s)” as usedherein. Pharmaceutically acceptable (i.e., non-toxic, physiologicallyacceptable) salts are preferred, although other salts are also useful,e.g., in isolation or purification steps which may be employed duringpreparation. Salts of the compounds of Formula I, II or III may beformed, for example, by reacting a compound with an amount of acid orbase, such as an equivalent amount, in a medium such as one in which thesalt precipitates or in an aqueous medium followed by lyophilization.

The compounds of the present invention which contain a basic moiety mayform salts with a variety of organic and inorganic acids. Exemplary acidaddition salts include acetates (such as those formed with acetic acidor trihaloacetic acid, for example, trifluoroacetic acid), adipates,alginates, ascorbates, aspartates, benzoates, benzenesulfonates,bisulfates, borates, butyrates, citrates, camphorates,camphorsulfonates, cyclopentanepropionates, digluconates,dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates,glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides(formed with hydrochloric acid), hydrobromides (formed with hydrogenbromide), hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates(formed with maleic acid), methanesulfonates (formed withmethanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates,oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates,picrates, pivalates, propionates, salicylates, succinates, sulfates(such as those formed with sulfuric acid), sulfonates (such as thosementioned herein), tartrates, thiocyanates, toluenesulfonates such astosylates, undecanoates, and the like.

The compounds of the present invention which contain an acidic moietymay form salts with a variety of organic and inorganic bases. Exemplarybasic salts include ammonium salts, alkali metal salts such as sodium,lithium, and potassium salts, alkaline earth metal salts such as calciumand magnesium salts, salts with organic bases (for example, organicamines) such as benzathines, dicyclohexylamines, hydrabamines (formedwith N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines,N-methyl-D-glucamides, t-butyl amines, and salts with amino acids suchas arginine, lysine and the like.

The stereochemical terms and conventions used in the specification areconsistent with those described in Pure & Appl. Chem 68:2193 (1996),unless otherwise indicated.

The term “enantiomeric excess” or “ee” refers to a measure for how muchof one enantiomer is present compared to the other. For a mixture of Rand S enantiomers, the percent enantiomeric excess is defined as|R−S|*100, where R and S are the respective mole or weight fractions ofenantiomers in a mixture such that R+S=1. With knowledge of the opticalrotation of a chiral substance, the percent enantiomeric excess isdefined as ([α]_(obs)/[α]_(max))*100, where [α]_(obs) is the opticalrotation of the mixture of enantiomers and [α]_(max) is the opticalrotation of the pure enantiomer. Determination of enantiomeric excess ispossible using a variety of analytical techniques, including NMRspectroscopy, chiral column chromatography or optical polarimetry.

The terms “enantiomerically pure” or “enantiopure” refer to a sample ofa chiral substance all of whose molecules (within the limits ofdetection) have the same chirality sense.

The terms “enantiomerically enriched” or “enantioenriched” refer to asample of a chiral substance whose enantiomeric ratio is greater than50:50. Enantiomerically enriched compounds may be enantiomerically pure.

The term “asymmetric carbon atom” refers to a carbon atom in a moleculeof an organic compound that is attached to four different atoms orgroups of atoms.

The term “predominantly” means in a ratio greater than 50:50.

The term “leaving group” or “LG” refers to an atom or group that becomesdetached from an atom or group in what is considered to be the residualor main part of the substrate in a specified reaction. In amide couplingreactions, exemplary leaving groups include —F, —Cl, —Br, —OC₆F₅ and thelike.

The term “isolated” for the purposes of the present invention designatesa material (e.g. a chemical compound or biological material such as anucleic acid or protein) that has been removed from its originalenvironment (the environment in which it is naturally present). Forexample, a polynucleotide present in the natural state in a plant or ananimal is not isolated, however the same polynucleotide separated fromthe adjacent nucleic acids in which it is naturally present, isconsidered “isolated”.

The term “purified”, as applied to biological materials does not requirethe material to be present in a form exhibiting absolute purity,exclusive of the presence of other compounds. It is rather a relativedefinition.

“Nucleic acid”, “nucleic acid molecule”, “oligonucleotide” and“polynucleotide” are used interchangeably and refer to the phosphateester polymeric form of ribonucleosides (adenosine, guanosine, uridineor cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine,deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), orany phosphoester analogs thereof, such as phosphorothioates andthioesters, in either single stranded form, or a double-stranded helix.Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. Theterm nucleic acid molecule, and in particular DNA or RNA molecule,refers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear or circularDNA molecules (e.g., restriction fragments), plasmids, supercoiled DNAand chromosomes. In discussing the structure of particulardouble-stranded DNA molecules, sequences may be described hereinaccording to the normal convention of giving only the sequence in the 5′to 3′ direction along the non-transcribed strand of DNA (i.e., thestrand having a sequence homologous to the mRNA). A “recombinant DNAmolecule” is a DNA molecule that has undergone a molecular biologicalmanipulation. DNA includes but is not limited to cDNA, genomic DNA,plasmid DNA, synthetic DNA, and semi-synthetic DNA.

The term “fragment” refers to a nucleotide sequence of reduced lengthrelative to the reference nucleic acid and comprising, over the commonportion, a nucleotide sequence identical to the reference nucleic acid.Such a nucleic acid fragment according to the invention may be, whereappropriate, included in a larger polynucleotide of which it is aconstituent. Such fragments comprise, or alternatively consist of,oligonucleotides ranging in length from at least 6, 8, 9, 10, 12, 15,18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60,63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720,900, 1000 or 1500 consecutive nucleotides of a nucleic acid according tothe invention.

As used herein, an “isolated nucleic acid fragment” refers to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A “gene” refers to a polynucleotide comprising nucleotides that encode afunctional molecule (e.g., a polypeptide or RNA), and includes cDNA andgenomic DNA nucleic acids. “Gene” also refers to a nucleic acid fragmentthat expresses a specific RNA, protein or polypeptide, includingregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and/or coding sequences that are not found together innature. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A chimericgene may comprise coding sequences derived from different sources and/orregulatory sequences derived from different sources. “Endogenous gene”refers to a native gene in its natural location in the genome of anorganism. A “foreign” gene or “heterologous” gene refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

The term “genome” includes chromosomal as well as mitochondrial,chloroplast and viral DNA or RNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., 1989 infra). Hybridization andwashing conditions are well known and exemplified in Sambrook et al. inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter11 and Table 11.1 therein (entirely incorporated herein by reference).The conditions of temperature and ionic strength determine the“stringency” of the hybridization.

Stringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC,0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5%SDS). Moderate stringency hybridization conditions correspond to ahigher T_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The term “complementary” is usedto describe the relationship between nucleotide bases that are capableof hybridizing to one another. For example, with respect to DNA,adenosine is complementary to thymine and cytosine is complementary toguanine. Accordingly, the present invention also includes isolatednucleic acid fragments that are complementary to the complete sequencesas disclosed or used herein as well as those substantially similarnucleic acid sequences.

In one embodiment of the invention, polynucleotides are detected byemploying hybridization conditions comprising a hybridization step atT_(m) of 55° C., and utilizing conditions as set forth above. In anotherembodiment, the T_(m) is 60° C.; in one embodiment, the T_(m) is 63° C.;in another embodiment, the T_(m) is 65° C.

Post-hybridization washes also determine stringency conditions. One setof conditions uses a series of washes starting with 6×SSC, 0.5% SDS atroom temperature for 15 minutes (min), then repeated with 2×SSC, 0.5%SDS at 45° C. for 30 minutes, and then repeated twice with 0.2×SSC, 0.5%SDS at 50° C. for 30 minutes. One set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS is increased to 60° C. Another set of highly stringentconditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA: RNA, DNA: RNA, DNA: DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8).

In one embodiment of the invention, polynucleotides are detected byemploying hybridization conditions comprising a hybridization step inless than 500 mM salt and at least 37 degrees Celsius, and a washingstep in 2×SSPE at least 63 degrees Celsius. In one embodiment, thehybridization conditions comprise less than 200 mM salt and at least 37degrees Celsius for the hybridization step. In a another embodiment, thehybridization conditions comprise 2×SSPE and 63 degrees Celsius for boththe hybridization and washing steps.

In another embodiment, the length for a hybridizable nucleic acid is atleast about 10 nucleotides. Preferably a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; morepreferably at least about 20 nucleotides; and most preferably the lengthis at least 30 nucleotides. Furthermore, the skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as length of theprobe.

The term “probe” refers to a single-stranded nucleic acid molecule thatcan base pair with a complementary single stranded target nucleic acidto form a double-stranded molecule.

As used herein, the term “oligonucleotide” refers to a short nucleicacid that is hybridizable to a genomic DNA molecule, a cDNA molecule, aplasmid DNA or an mRNA molecule. Oligonucleotides can be labeled, e.g.,with ³²P-nucleotides or nucleotides to which a label, such as biotin,has been covalently conjugated. A labeled oligonucleotide can be used asa probe to detect the presence of a nucleic acid. Oligonucleotides (oneor both of which may be labeled) can be used as PCR primers, either forcloning full length or a fragment of a nucleic acid, or to detect thepresence of a nucleic acid. An oligonucleotide can also be used to forma triple helix with a DNA molecule. Generally, oligonucleotides areprepared synthetically, preferably on a nucleic acid synthesizer.Accordingly, oligonucleotides can be prepared with non-naturallyoccurring phosphoester analog bonds, such as thioester bonds, etc.

A “primer” refers to an oligonucleotide that hybridizes to a targetnucleic acid sequence to create a double stranded nucleic acid regionthat can serve as an initiation point for DNA synthesis under suitableconditions. Such primers may be used in a polymerase chain reaction.

“Polymerase chain reaction” is abbreviated PCR and refers to an in vitromethod for enzymatically amplifying specific nucleic acid sequences. PCRinvolves a repetitive series of temperature cycles with each cyclecomprising three stages: denaturation of the template nucleic acid toseparate the strands of the target molecule, annealing a single strandedPCR oligonucleotide primer to the template nucleic acid, and extensionof the annealed primer(s) by DNA polymerase. PCR provides a means todetect the presence of the target molecule and, under quantitative orsemi-quantitative conditions, to determine the relative amount of thattarget molecule within the starting pool of nucleic acids.

“Reverse transcription-polymerase chain reaction” is abbreviated RT-PCRand refers to an in vitro method for enzymatically producing a targetcDNA molecule or molecules from an RNA molecule or molecules, followedby enzymatic amplification of a specific nucleic acid sequence orsequences within the target cDNA molecule or molecules as describedabove. RT-PCR also provides a means to detect the presence of the targetmolecule and, under quantitative or semi-quantitative conditions, todetermine the relative amount of that target molecule within thestarting pool of nucleic acids.

A DNA “coding sequence” refers to a double-stranded DNA sequence thatencodes a polypeptide and can be transcribed and translated into apolypeptide in a cell in vitro or in vivo when placed under the controlof appropriate regulatory sequences. “Suitable regulatory sequences”refers to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing site, effectorbinding site and stem-loop structure. The boundaries of the codingsequence are determined by a start codon at the 5′ (amino) terminus anda translation stop codon at the 3′ (carboxyl) terminus. A codingsequence can include, but is not limited to, prokaryotic sequences, cDNAfrom mRNA, genomic DNA sequences, and even synthetic DNA sequences. Ifthe coding sequence is intended for expression in a eukaryotic cell, apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

“Open reading frame” is abbreviated ORF and refers to a length ofnucleic acid sequence, either DNA, cDNA or RNA, that comprises atranslation start signal or initiation codon, such as an ATG or AUG, anda termination codon and can be potentially translated into a polypeptidesequence.

The term “head-to-head” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-head orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 5′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds away from the 5′ end ofthe other polynucleotide. The term “head-to-head” may be abbreviated(5′)-to-(5′) and may also be indicated by the symbols (← →) or(3′←5′5′→3′).

The term “tail-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a tail-to-tail orientation when the 3′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds toward the otherpolynucleotide. The term “tail-to-tail” may be abbreviated (3′)-to-(3′)and may also be indicated by the symbols (→ ←) or (5′→3′3′←5′).

The term “head-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-tail orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds in the same directionas that of the other polynucleotide. The term “head-to-tail” may beabbreviated (5′)-to-(3′) and may also be indicated by the symbols (→ →)or (5′→3′5′→3′).

The term “downstream” refers to a nucleotide sequence that is located 3′to a reference nucleotide sequence. In particular, downstream nucleotidesequences generally relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to a reference nucleotide sequence. In particular, upstream nucleotidesequences generally relate to sequences that are located on the 5′ sideof a coding sequence or starting point of transcription. For example,most promoters are located upstream of the start site of transcription.

The terms “restriction endonuclease” and “restriction enzyme” are usedinterchangeably and refer to an enzyme that binds and cuts within aspecific nucleotide sequence within double stranded DNA.

“Homologous recombination” refers to the insertion of a foreign DNAsequence into another DNA molecule, e.g., insertion of a vector in achromosome. Preferably, the vector targets a specific chromosomal sitefor homologous recombination. For specific homologous recombination, thevector will contain sufficiently long regions of homology to sequencesof the chromosome to allow complementary binding and incorporation ofthe vector into the chromosome. Longer regions of homology, and greaterdegrees of sequence similarity, may increase the efficiency ofhomologous recombination.

Several methods known in the art may be used to propagate apolynucleotide according to the invention. Once a suitable host systemand growth conditions are established, recombinant expression vectorscan be propagated and prepared in quantity. As described herein, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

A “vector” refers to any vehicle for the cloning of and/or transfer of anucleic acid into a host cell. A vector may be a replicon to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” refers to any genetic element(e.g., plasmid, phage, cosmid, chromosome, virus) that functions as anautonomous unit of DNA replication in vivo, i.e., capable of replicationunder its own control. The term “vector” includes both viral andnonviral vehicles for introducing the nucleic acid into a cell in vitro,ex vivo or in vivo. A large number of vectors known in the art may beused to manipulate nucleic acids, incorporate response elements andpromoters into genes, etc. Possible vectors include, for example,plasmids or modified viruses including, for example bacteriophages suchas lambda derivatives, or plasmids such as pBR322 or pUC plasmidderivatives, or the Bluescript vector. For example, the insertion of theDNA fragments corresponding to response elements and promoters into asuitable vector can be accomplished by ligating the appropriate DNAfragments into a chosen vector that has complementary cohesive termini.Alternatively, the ends of the DNA molecules may be enzymaticallymodified or any site may be produced by ligating nucleotide sequences(linkers) into the DNA termini. Such vectors may be engineered tocontain selectable marker genes that provide for the selection of cellsthat have incorporated the marker into the cellular genome. Such markersallow identification and/or selection of host cells that incorporate andexpress the proteins encoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in awide variety of gene delivery applications in cells, as well as livinganimal subjects. Viral vectors that can be used include but are notlimited to retrovirus, adeno-associated virus, pox, baculovirus,vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, andcaulimovirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers. In addition to a nucleic acid, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(transfer to which tissues, duration of expression, etc.).

The term “plasmid” refers to an extra-chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A “cloning vector” refers to a “replicon,” which is a unit length of anucleic acid, preferably DNA, that replicates sequentially and whichcomprises an origin of replication, such as a plasmid, phage or cosmid,to which another nucleic acid segment may be attached so as to bringabout the replication of the attached segment. Cloning vectors may becapable of replication in one cell type and expression in another(“shuttle vector”).

The term “expression vector” refers to a vector, plasmid or vehicledesigned to enable the expression of an inserted nucleic acid sequencefollowing transformation into the host. The cloned gene, i.e., theinserted nucleic acid sequence, is usually placed under the control ofcontrol elements such as a promoter, a minimal promoter, an enhancer, orthe like. Initiation control regions or promoters, which are useful todrive expression of a nucleic acid in the desired host cell are numerousand familiar to those skilled in the art. Virtually any promoter capableof driving these genes is suitable for the present invention includingbut not limited to: viral promoters, bacterial promoters, animalpromoters, mammalian promoters, synthetic promoters, constitutivepromoters, tissue specific promoter, developmental specific promoters,inducible promoters, light regulated promoters; CYC1, HIS3, GAL1, GAL4,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI,alkaline phosphatase promoters (useful for expression in Saccharomyces);AOX1 promoter (useful for expression in Pichia); β-lactamase, lac, ara,tet, trp, lP_(L), lP_(R), T7, tac, and trc promoters (useful forexpression in Escherichia coli); light regulated-, seed specific-,pollen specific-, ovary specific-, pathogenesis or disease related-,cauliflower mosaic virus 35S, CMV 35S minimal, cassava vein mosaic virus(CsVMV), chlorophyll a/b binding protein, ribulose 1,5-bisphosphatecarboxylase, shoot-specific, root specific, chitinase, stress inducible,rice tungro bacilliform virus, plant super-promoter, potato leucineaminopeptidase, nitrate reductase, mannopine synthase, nopalinesynthase, ubiquitin, zein protein, and anthocyanin promoters (useful forexpression in plant cells); animal and mammalian promoters known in theart include, but are not limited to, the SV40 early (SV40e) promoterregion, the promoter contained in the 3′ long terminal repeat (LTR) ofRous sarcoma virus (RSV), the promoters of the E1A or major latepromoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV)early promoter, the herpes simplex virus (HSV) thymidine kinase (TK)promoter, a baculovirus IE1 promoter, an elongation factor 1 alpha (EF1)promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc)promoter, an albumin promoter, the regulatory sequences of the mousemetallothionein-L promoter and transcriptional control regions, theubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like),the promoters of the intermediate filaments (desmin, neurofilaments,keratin, GFAP, and the like), the promoters of therapeutic genes (of theMDR, CFTR or factor VIII type, and the like), pathogenesis or diseaserelated-promoters, and promoters that exhibit tissue specificity andhave been utilized in transgenic animals, such as the elastase I genecontrol region which is active in pancreatic acinar cells; insulin genecontrol region active in pancreatic beta cells, immunoglobulin genecontrol region active in lymphoid cells, mouse mammary tumor viruscontrol region active in testicular, breast, lymphoid and mast cells;albumin gene, Apo AI and Apo AII control regions active in liver,alpha-fetoprotein gene control region active in liver, alpha1-antitrypsin gene control region active in the liver, beta-globin genecontrol region active in myeloid cells, myelin basic protein genecontrol region active in oligodendrocyte cells in the brain, myosinlight chain-2 gene control region active in skeletal muscle, andgonadotropic releasing hormone gene control region active in thehypothalamus, pyruvate kinase promoter, villin promoter, promoter of thefatty acid binding intestinal protein, promoter of the smooth musclecell α-actin, and the like. In addition, these expression sequences maybe modified by addition of enhancer or regulatory sequences and thelike.

Vectors may be introduced into the desired host cells by methods knownin the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963-967(1992); Wu et al., J. Biol. Chem. 263:14621-14624 (1988); and Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

A polynucleotide according to the invention can also be introduced invivo by lipofection. For the past decade, there has been increasing useof liposomes for encapsulation and transfection of nucleic acids invitro. Synthetic cationic lipids designed to limit the difficulties anddangers encountered with liposome-mediated transfection can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Felgner et al., Proc. Natl. Acad. Sci. USA. 84:7413 (1987); Mackey etal., Proc. Natl. Acad. Sci. USA 85:8027-8031 (1988); and Ulmer et al.,Science 259:1745-1748 (1993)). The use of cationic lipids may promoteencapsulation of negatively charged nucleic acids, and also promotefusion with negatively charged cell membranes (Felgner et al., Science337:387-388 (1989)). Particularly useful lipid compounds andcompositions for transfer of nucleic acids are described in WO95/18863,WO96/17823 and U.S. Pat. No. 5,459,127. The use of lipofection tointroduce exogenous genes into the specific organs in vivo has certainpractical advantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly preferred in a tissuewith cellular heterogeneity, such as pancreas, liver, kidney, and thebrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting (Mackey et al. 1988, supra). Targeted peptides,e.g., hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as a naked DNA plasmid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., Hum. Gene Ther. 3:147-154 (1992); and Wu et al., J. Biol. Chem.262:4429-4432 (1987)).

The term “transfection” refers to the uptake of exogenous orheterologous RNA or DNA by a cell. A cell has been “transfected” byexogenous or heterologous RNA or DNA when such RNA or DNA has beenintroduced inside the cell. A cell has been “transformed” by exogenousor heterologous RNA or DNA when the transfected RNA or DNA effects aphenotypic change. The transforming RNA or DNA can be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

In addition, the recombinant vector comprising a polynucleotideaccording to the invention may include one or more origins forreplication in the cellular hosts in which their amplification or theirexpression is sought, markers or selectable markers.

The term “selectable marker” refers to an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,resistance to a herbicide, calorimetric markers, enzymes, fluorescentmarkers, and the like, wherein the effect is used to track theinheritance of a nucleic acid of interest and/or to identify a cell ororganism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like.

The term “reporter gene” refers to a nucleic acid encoding anidentifying factor that is able to be identified based upon the reportergene's effect, wherein the effect is used to track the inheritance of anucleic acid of interest, to identify a cell or organism that hasinherited the nucleic acid of interest, and/or to measure geneexpression induction or transcription. Examples of reporter genes knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable marker genes may also beconsidered reporter genes.

“Promoter and “promoter sequence” are used interchangeably and refer toa DNA sequence capable of controlling the expression of a codingsequence or functional RNA. In general, a coding sequence is located 3′to a promoter sequence. Promoters may be derived in their entirety froma native gene, or be composed of different elements derived fromdifferent promoters found in nature, or even comprise synthetic DNAsegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental or physiological conditions. Promoters thatcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters.” Promoters that cause agene to be expressed in a specific cell type are commonly referred to as“cell-specific promoters” or “tissue-specific promoters.” Promoters thatcause a gene to be expressed at a specific stage of development or celldifferentiation are commonly referred to as “developmentally-specificpromoters” or “cell differentiation-specific promoters.” Promoters thatare induced and cause a gene to be expressed following exposure ortreatment of the cell with an agent, biological molecule, chemical,ligand, light, or the like that induces the promoter are commonlyreferred to as “inducible promoters” or “regulatable promoters.” It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The promoter sequence is typically bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced (if the coding sequence contains introns) and translated intothe protein encoded by the coding sequence.

“Transcriptional and translational control sequences” refer to DNAregulatory sequences, such as promoters, enhancers, terminators, and thelike, that provide for the expression of a coding sequence in a hostcell. In eukaryotic cells, polyadenylation signals are controlsequences.

The term “response element” refers to one or more cis-acting DNAelements which confer responsiveness on a promoter mediated throughinteraction with the DNA-binding domains of a transcription factor,e.g., the DNA binding domain of the first hybrid protein. This DNAelement may be either palindromic (perfect or imperfect) in its sequenceor composed of sequence motifs or half sites separated by a variablenumber of nucleotides. The half sites can be similar or identical andarranged as either direct or inverted repeats or as a single half siteor multimers of adjacent half sites in tandem. The response element maycomprise a minimal promoter isolated from different organisms dependingupon the nature of the cell or organism into which the response elementwill be incorporated. The DNA binding domain of the first hybrid proteinbinds, in the presence or absence of a ligand, to the DNA sequence of aresponse element to initiate or suppress transcription of downstreamgene(s) under the regulation of this response element. Examples of DNAsequences for response elements of the natural ecdysone receptorinclude: RRGG/TTCANTGAC/ACYY (SEQ ID NO: 16) (see Cherbas et. al., GenesDev. 5:120-131 (1991)); AGGTCAN_((n))AGGTCA, where N_((n)) can be one ormore spacer nucleotides (SEQ ID NO: 17) (see D'Avino et al., Mol. Cell.Endocrinol. 113:1-9 (1995)); and GGGTTGAATGAATTT (SEQ ID NO: 18) (seeAntoniewski et al., Mol. Cell Biol. 14:4465-4474 (1994)).

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression” as used herein refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from anucleic acid or polynucleotide. Expression may also refer to translationof mRNA into a protein or polypeptide.

The terms “cassette,” “expression cassette” and “gene expressioncassette” refer to a segment of DNA that can be inserted into a nucleicacid or polynucleotide at specific restriction sites or by homologousrecombination. The segment of DNA comprises a polynucleotide thatencodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. “Transformationcassette” refers to a specific vector comprising a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.Cassettes, expression cassettes, gene expression cassettes andtransformation cassettes of the invention may also comprise elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

For purposes of this invention, the term “gene switch” refers to thecombination of a response element associated with a promoter, and anEcR-based system which in the presence of one or more ligands, modulatesthe expression of a gene into which the response element and promoterare incorporated.

The terms “modulate” and “modulates” mean to induce, reduce or inhibitnucleic acid or gene expression, resulting in the respective induction,reduction or inhibition of protein or polypeptide production.

The plasmids or vectors according to the invention may further compriseat least one promoter suitable for driving expression of a gene in ahost cell.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor 1 (EF1) enhancer, yeast enhancers, viral geneenhancers, and the like.

Termination control regions, i.e., terminator or polyadenylationsequences, may also be derived from various genes native to thepreferred hosts. Optionally, a termination site may be unnecessary,however, it is most preferred if included. In one embodiment of theinvention, the termination control region may be comprised or be derivedfrom a synthetic sequence, synthetic polyadenylation signal, an SV40late polyadenylation signal, an SV40 polyadenylation signal, a bovinegrowth hormone (BGH) polyadenylation signal, viral terminator sequences,or the like.

The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)”refer to DNA sequences located downstream (3′) of a coding sequence andmay comprise polyadenylation [poly(A)] recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor.

“Regulatory region” refers to a nucleic acid sequence that regulates theexpression of a second nucleic acid sequence. A regulatory region mayinclude sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin that are responsible for expressing differentproteins or even synthetic proteins (a heterologous region). Inparticular, the sequences can be sequences of prokaryotic, eukaryotic,or viral genes or derived sequences that stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, promoters, enhancers, transcriptionaltermination sequences, and signal sequences which direct the polypeptideinto the secretory pathways of the target cell.

A regulatory region from a “heterologous source” refers to a regulatoryregion that is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but which are designed by one having ordinary skill inthe art.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or thecoding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA,or other RNA that is not translated yet has an effect on cellularprocesses.

“Polypeptide,” “peptide” and “protein” are used interchangeably andrefer to a polymeric compound comprised of covalently linked amino acidresidues. Amino acids have the following general structure:

An “isolated polypeptide,” “isolated peptide” or “isolated protein”refers to a polypeptide or protein that is substantially free of thosecompounds that are normally associated therewith in its natural state(e.g., other proteins or polypeptides, nucleic acids, carbohydrates,lipids). “Isolated” is not meant to exclude artificial or syntheticmixtures with other compounds, or the presence of impurities which donot interfere with biological activity, and which may be present, forexample, due to incomplete purification, addition of stabilizers, orcompounding into a pharmaceutically acceptable preparation.

A “substitution mutant polypeptide” or a “substitution mutant” will beunderstood to mean a mutant polypeptide comprising a substitution of atleast one (1) wild-type or naturally occurring amino acid with adifferent amino acid relative to the wild-type or naturally occurringpolypeptide. A substitution mutant polypeptide may comprise only one (1)wild-type or naturally occurring amino acid substitution and may bereferred to as a “point mutant” or a “single point mutant” polypeptide.Alternatively, a substitution mutant polypeptide may comprise asubstitution of two (2) or more wild-type or naturally occurring aminoacids with 2 or more amino acids relative to the wild-type or naturallyoccurring polypeptide. According to the invention, a Group H nuclearreceptor ligand binding domain polypeptide comprising a substitutionmutation comprises a substitution of at least one (1) wild-type ornaturally occurring amino acid with a different amino acid relative tothe wild-type or naturally occurring Group H nuclear receptor ligandbinding domain polypeptide.

Wherein the substitution mutant polypeptide comprises a substitution oftwo (2) or more wild-type or naturally occurring amino acids, thissubstitution may comprise either an equivalent number of wild-type ornaturally occurring amino acids deleted for the substitution, i.e., 2wild-type or naturally occurring amino acids replaced with 2non-wild-type or non-naturally occurring amino acids, or anon-equivalent number of wild-type amino acids deleted for thesubstitution, i.e., 2 wild-type amino acids replaced with 1non-wild-type amino acid (a substitution+deletion mutation), or 2wild-type amino acids replaced with 3 non-wild-type amino acids (asubstitution+insertion mutation).

Substitution mutants may be described using an abbreviated nomenclaturesystem to indicate the amino acid residue and number replaced within thereference polypeptide sequence and the new substituted amino acidresidue. For example, a substitution mutant in which the twentieth(20^(th)) amino acid residue of a polypeptide is substituted may beabbreviated as “x20z”, wherein “x” is the amino acid to be replaced,“20” is the amino acid residue position or number within thepolypeptide, and “z” is the new substituted amino acid. Therefore, asubstitution mutant abbreviated interchangeably as “E20A” or “Glu20Ala”indicates that the mutant comprises an alanine residue (commonlyabbreviated in the art as “A” or “Ala”) in place of the glutamic acid(commonly abbreviated in the art as “E” or “Glu”) at position 20 of thepolypeptide.

A substitution mutation may be made by any technique for mutagenesisknown in the art, including but not limited to, in vitro site-directedmutagenesis (Hutchinson et al., J. Biol. Chem. 253:6551 (1978); Zolleret al., DNA 3:479-488 (1984); Oliphant et al., Gene 44:177 (1986);Hutchinson et al., Proc. Natl. Acad. Sci. USA 83:710 (1986)), use ofTAB® linkers (Pharmacia), restriction endonuclease digestion/fragmentdeletion and substitution, PCR-mediated/oligonucleotide-directedmutagenesis, and the like. PCR-based techniques are preferred forsite-directed mutagenesis (see Higuchi, 1989, “Using PCR to EngineerDNA”, in PCR Technology: Principles and Applications for DNAAmplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

“Fragment” of a polypeptide according to the invention refers to apolypeptide whose amino acid sequence is shorter than that of thereference polypeptide and which comprises, over the entire portion withthese reference polypeptides, an identical amino acid sequence. Suchfragments may, where appropriate, be included in a larger polypeptide ofwhich they are a part. Such fragments of a polypeptide according to theinvention may have a length of at least 2, 3, 4, 5, 6, 8, 10, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200,240, or 300 amino acids.

A “variant” of a polypeptide or protein refers to any analogue,fragment, derivative, or mutant which is derived from a polypeptide orprotein and which retains at least one biological property of thepolypeptide or protein. Different variants of the polypeptide or proteinmay exist in nature. These variants may be allelic variationscharacterized by differences in the nucleotide sequences of thestructural gene coding for the protein, or may involve differentialsplicing or post-translational modification. The skilled artisan canproduce variants having single or multiple amino acid substitutions,deletions, additions, or replacements. These variants may include, interalia: (a) variants in which one or more amino acid residues aresubstituted with conservative or non-conservative amino acids, (b)variants in which one or more amino acids are added to the polypeptideor protein, (c) variants in which one or more of the amino acidsincludes a substituent group, and (d) variants in which the polypeptideor protein is fused with another polypeptide such as serum albumin. Thetechniques for obtaining these variants, including genetic(suppressions, deletions, mutations, etc.), chemical, and enzymatictechniques, are known to persons having ordinary skill in the art. Avariant polypeptide preferably comprises at least about 14 amino acids.

The term “homology” refers to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown to the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions that form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s) and size determination of thedigested fragments.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., Cell 50:667 (1987)). Such proteins (and their encoding genes)have sequence homology, as reflected by their high degree of sequencesimilarity. However, in common usage and in the present application, theterm “homologous,” when modified with an adverb such as “highly,” mayrefer to sequence similarity and not a common evolutionary origin.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., Cell 50:667 (1987)). In oneembodiment, two DNA sequences are “substantially homologous” or“substantially similar” when at least about 50% (preferably at leastabout 75%, and most preferably at least about 90 or 95%) of thenucleotides match over the defined length of the DNA sequences.Sequences that are substantially homologous can be identified bycomparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart (see e.g., Sambrook et al., 1989, supra.).

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate alteration of gene expression by antisense or co-suppressiontechnology. “Substantially similar” also refers to modifications of thenucleic acid fragments of the present invention such as deletion orinsertion of one or more nucleotide bases that do not substantiallyaffect the functional properties of the resulting transcript. It istherefore understood that the invention encompasses more than thespecific exemplary sequences. Each of the proposed modifications is wellwithin the routine skill in the art, as is determination of retention ofbiological activity of the encoded products.

Moreover, the skilled artisan recognizes that substantially similarsequences encompassed by this invention are also defined by theirability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65°C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), withthe sequences exemplified herein. Substantially similar nucleic acidfragments of the present invention are those nucleic acid fragmentswhose DNA sequences are at least 70% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Nucleic acid fragments ofthe present invention include those nucleic acid fragments whose DNAsequences are at least 80% identical to the DNA sequence of the nucleicacid fragments reported herein. Other nucleic acid fragments are atleast 90% identical to the DNA sequence of the nucleic acid fragmentsreported herein. Other nucleic acid fragments are at least 95% identicalto the DNA sequence of the nucleic acid fragments reported herein.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than about 40% of the amino acidsare identical, or greater than 60% are similar (functionally identical).Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al., J. Mol. Biol. 215:403-410 (1993)); see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence.

The term “percent identity,” as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Methods todetermine identity are designed to give the best match between thesequences tested. Methods to determine identity and similarity arecodified in publicly available computer programs. Sequence alignmentsand percent identity calculations may be performed using the Megalignprogram of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Multiple alignment of the sequences may be performedusing the Clustal method of alignment (Higgins et al., CABIOS. 5:151-153(1989)) with the default parameters (GAP PENALTY=10, GAP LENGTHPENALTY=10). Default parameters for pairwise alignments using theClustal method may be selected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include but is not limited to the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410(1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715USA). Within the context of this application it will be understood thatwhere sequence analysis software is used for analysis, that the resultsof the analysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters which originally load with thesoftware when first initialized.

“Chemically synthesized,” as related to a sequence of DNA, means thatthe component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

As used herein, two or more individually operable gene regulationsystems are said to be “orthogonal” when; a) modulation of each of thegiven systems by its respective ligand, at a chosen concentration,results in a measurable change in the magnitude of expression of thegene of that system, and b) the change is statistically significantlydifferent than the change in expression of all other systemssimultaneously operable in the cell, tissue, or organism, regardless ofthe simultaneity or sequentially of the actual modulation. Preferably,modulation of each individually operable gene regulation system effectsa change in gene expression at least 2-fold greater than all otheroperable systems in the cell, tissue, or organism. More preferably, thechange is at least 5-fold greater. Even more preferably, the change isat least 10-fold greater. Still more preferably, the change is at least100 fold greater. Even still more preferably, the change is at least500-fold greater. Ideally, modulation of each of the given systems byits respective ligand at a chosen concentration results in a measurablechange in the magnitude of expression of the gene of that system and nomeasurable change in expression of all other systems operable in thecell, tissue, or organism. In such cases the multiple inducible generegulation system is said to be “fully orthogonal.” The presentinvention is useful to search for orthogonal ligands and orthogonalreceptor-based gene expression systems such as those described in US2002/0110861 A1, which is incorporated herein by reference in itsentirety.

The term “modulate” means the ability of a given ligand/receptor complexto induce or suppress the transactivation of an exogenous gene.

The term “exogenous gene” means a gene foreign to the subject, that is,a gene which is introduced into the subject through a transformationprocess, an unmutated version of an endogenous mutated gene or a mutatedversion of an endogenous unmutated gene. The method of transformation isnot critical to this invention and may be any method suitable for thesubject known to those in the art. For example, transgenic plants areobtained by regeneration from the transformed cells. Numeroustransformation procedures are known from the literature such asagroinfection using Agrobacterium tumefaciens or its T₁ plasmid,electroporation, microinjection of plant cells and protoplasts, andmicroprojectile transformation. Complementary techniques are known fortransformation of animal cells and regeneration of such transformedcells in transgenic animals. Exogenous genes can be either natural orsynthetic genes and therapeutic genes which are introduced into thesubject in the form of DNA or RNA which may function through a DNAintermediate such as by reverse transcriptase. Such genes can beintroduced into target cells, directly introduced into the subject, orindirectly introduced by the transfer of transformed cells, e.g.autologous cells, into the subject. The term “therapeutic gene” means agene which imparts a beneficial function to the host cell in which suchgene is expressed.

The term “ecdysone receptor complex” generally refers to a heterodimericprotein complex consisting of two members of the steroid receptorfamily, ecdysone receptor (“EcR”) and ultraspiracle (“USP”) proteins(see Yao et al., Nature 366:476-479 (1993)); Yao et al., Cell 71:63-72(1992)). The functional ecdysteroid receptor complex may also includeadditional protein(s) such as immunophilins. Additional members of thesteroid receptor family of proteins, known as transcriptional factors(such as DHR38, betaFTZ-1 or other insect homologs), may also be liganddependent or independent partners for EcR and/or USP. The ecdysonereceptor complex can also be a heterodimer of ecdysone receptor proteinand the vertebrate homolog of ultraspiracle protein, retinoicacid-X-receptor (“RXR”) protein. Homodimer complexes of the ecdysonereceptor protein or USP may also be functional under some circumstances.

An ecdysteroid receptor complex can be activated by an activeecdysteroid or non-steroidal ligand bound to one of the proteins of thecomplex, inclusive of EcR, but not excluding other proteins of thecomplex.

The ecdysone receptor complex includes proteins which are members of thesteroid receptor superfamily wherein all members are characterized bythe presence of an amino-terminal transactivation domain, a DNA bindingdomain (“DBD”), and a ligand binding domain (“LBD”) separated by a hingeregion. Some members of the family may also have another transactivationdomain on the carboxy-terminal side of the LBD. The DBD is characterizedby the presence of two cysteine zinc fingers between which are two aminoacid motifs, the P-box and the D-box, which confer specificity forecdysone response elements. These domains may be either native,modified, or chimeras of different domains of heterologous receptorproteins.

The DNA sequences making up the exogenous gene, the response element,and the ecdysone receptor complex may be incorporated intoarchaebacteria, procaryotic cells such as Escherichia coli, Bacillussubtilis, or other enterobacteria, or eucaryotic cells such as plant oranimal cells. However, because many of the proteins expressed by thegene are processed incorrectly in bacteria, eucaryotic cells arepreferred. The cells may be in the form of single cells or multicellularorganisms. The nucleotide sequences for the exogenous gene, the responseelement, and the receptor complex can also be incorporated as RNAmolecules, preferably in the form of functional viral RNAs such astobacco mosaic virus. Of the eucaryotic cells, vertebrate cells arepreferred because they naturally lack the molecules which conferresponses to the ligands of this invention for the ecdysone receptor. Asa result, they are “substantially insensitive” to the ligands of thisinvention. Thus, the ligands of this invention will have negligiblephysiological or other effects on transformed cells, or the wholeorganism. Therefore, cells can grow and express the desired product,substantially unaffected by the presence of the ligand itself.

The term “subject” means an intact insect, plant or animal or a cellfrom an insect, plant or animal. It is also anticipated that the ligandswill work equally well when the subject is a fungus or yeast. When thesubject is an intact animal, preferably the animal is a vertebrate, mostpreferably a mammal.

The diacylhydrazine ligands of Formula I and chiral diacylhydrazineligands of Formula II or III of the present invention, when used withthe ecdysone receptor complex which in turn is bound to the responseelement linked to an exogenous gene, provide the means for externaltemporal regulation of expression of the exogenous gene. The order inwhich the various components bind to each other, that is, ligand toreceptor complex and receptor complex to response element, is notcritical. Typically, modulation of expression of the exogenous gene isin response to the binding of the ecdysone receptor complex to aspecific control, or regulatory, DNA element. The ecdysone receptorprotein, like other members of the steroid receptor family, possesses atleast three domains, a transactivation domain, a DNA binding domain, anda ligand binding domain. This receptor, like a subset of the steroidreceptor family, also possesses less well-defined regions responsiblefor heterodimerization properties. Binding of the ligand to the ligandbinding domain of ecdysone receptor protein, after heterodimerizationwith USP or RXR protein, enables the DNA binding domains of theheterodimeric proteins to bind to the response element in an activatedform, thus resulting in expression or suppression of the exogenous gene.This mechanism does not exclude the potential for ligand binding toeither EcR or USP, and the resulting formation of active homodimercomplexes (e.g. EcR+EcR or USP+USP). Preferably, one or more of thereceptor domains can be varied producing a chimeric gene switch.Typically, one or more of the three domains may be chosen from a sourcedifferent than the source of the other domains so that the chimericreceptor is optimized in the chosen host cell or organism fortransactivating activity, complementary binding of the ligand, andrecognition of a specific response element. In addition, the responseelement itself can be modified or substituted with response elements forother DNA binding protein domains such as the GAL-4 protein from yeast(see Sadowski et al., Nature 335:563-564 (1988) or LexA protein from E.coli (see Brent et al., Cell 43:729-736 (1985)) to accommodate chimericecdysone receptor complexes. Another advantage of chimeric systems isthat they allow choice of a promoter used to drive the exogenous geneaccording to a desired end result. Such double control can beparticularly important in areas of gene therapy, especially whencytotoxic proteins are produced, because both the timing of expressionas well as the cells wherein expression occurs can be controlled. Whenexogenous genes, operatively linked to a suitable promoter, areintroduced into the cells of the subject, expression of the exogenousgenes is controlled by the presence of the ligand of this invention.Promoters may be constitutively or inducibly regulated or may betissue-specific (that is, expressed only in a particular type of cell)or specific to certain developmental stages of the organism.

Numerous genomic and cDNA nucleic acid sequences coding for a variety ofpolypeptides are well known in the art. Exogenous genetic materialuseful with the ligands of this invention include genes that encodebiologically active proteins of interest, such as, for example,secretory proteins that can be released from a cell; enzymes that canmetabolize a substrate from a toxic substance to a non-toxic substance,or from an inactive substance to an active substance; regulatoryproteins; cell surface receptors; and the like. Useful genes alsoinclude genes that encode blood clotting factors, hormones such asinsulin, parathyroid hormone, luteinizing hormone releasing factor,alpha and beta seminal inhibins, and human growth hormone; genes thatencode proteins such as enzymes, the absence of which leads to theoccurrence of an abnormal state; genes encoding cytokines or lymphokinessuch as interferons, granulocytic macrophage colony stimulating factor,colony stimulating factor-1, tumor necrosis factor, and erythropoietin;genes encoding inhibitor substances such as alpha₁-antitrypsin, genesencoding substances that function as drugs such as diphtheria andcholera toxins; and the like. Useful genes also include those useful forcancer therapies and to treat genetic disorders. Those skilled in theart have access to nucleic acid sequence information for virtually allknown genes and can either obtain the nucleic acid molecule directlyfrom a public depository, the institution that published the sequence,or employ routine methods to prepare the molecule.

In one embodiment, the exogenous gene is the hIL-12 gene under controlof the RheoSwitch™ Therapeutic System (RTS), transfected ex vivo with anadenovirus into autologous dendritic cells.

For gene therapy use, the ligands described herein may be administeredalone or as part of a pharmaceutical composition comprising apharmaceutically acceptable carrier. In one embodiment, thepharmaceutical composition are in the form of solutions, suspensions,tablets, capsules, ointments, elixirs, or injectable compositions.

Pharmaceutically acceptable carriers include fillers such assaccharides, for example lactose or sucrose, mannitol or sorbitol,cellulose preparations and/or calcium phosphates, for example tricalciumphosphate or calcium hydrogen phosphate, as well as binders such asstarch paste, using, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone. If desired, disintegrating agents may be addedsuch as the above-mentioned starches and also carboxymethyl-starch,cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate. Auxiliaries are flow-regulating agentsand lubricants, for example, silica, talc, stearic acid or saltsthereof, such as magnesium stearate or calcium stearate, and/orpolyethylene glycol. In one embodiment, dragee cores are provided withsuitable coatings which, if desired, are resistant to gastric juices.For this purpose, concentrated saccharide solutions may be used, whichmay optionally contain gum arabic, talc, polyvinyl pyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate orhydroxypropylmethyl-cellulose phthalate, are used. Dye stuffs orpigments may be added to the tablets or dragee coatings, for example,for identification or in order to characterize combinations of activecompound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules ornanoparticles which may optionally be mixed with fillers such aslactose, binders such as starches, and/or lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In one embodiment, theis dissolved or suspended in suitable liquids, such as fatty oils, orliquid paraffin, optionally with stabilizers.

Fatty oils may comprise mono-, di- or triglycerides. Mono-, di- andtriglycerides include those that are derived from C₆, C₈, C₁₀, C₁₂, C₁₄,C₁₆, C₁₈, C₂₀ and C₂₂ acids. Exemplary diglycerides include, inparticular, diolein, dipalmitolein, and mixed caprylin-caprindiglycerides. Preferred triglycerides include vegetable oils, fish oils,animal fats, hydrogenated vegetable oils, partially hydrogenatedvegetable oils, synthetic triglycerides, modified triglycerides,fractionated triglycerides, medium and long-chain triglycerides,structured triglycerides, and mixtures thereof. Exemplary triglyceridesinclude: almond oil; babassu oil; borage oil; blackcurrant seed oil;canola oil; castor oil; coconut oil; corn oil; cottonseed oil; eveningprimrose oil; grapeseed oil; groundnut oil; mustard seed oil; olive oil;palm oil; palm kernel oil; peanut oil; rapeseed oil; safflower oil;sesame oil; shark liver oil; soybean oil; sunflower oil; hydrogenatedcastor oil; hydrogenated coconut oil; hydrogenated palm oil;hydrogenated soybean oil; hydrogenated vegetable oil; hydrogenatedcottonseed and castor oil; partially hydrogenated soybean oil; partiallysoy and cottonseed oil; glyceryl tricaproate; glyceryl tricaprylate;glyceryl tricaprate; glyceryl triundecanoate; glyceryl trilaurate;glyceryl trioleate; glyceryl trilinoleate; glyceryl trilinolenate;glyceryl tricaprylate/caprate; glyceryl tricaprylate/caprate/laurate;glyceryl tricaprylate/caprate/linoleate; and glyceryltricaprylate/caprate/stearate.

In one embodiment, the triglyceride is the medium chain triglycerideavailable under the trade name LABRAFAC CC. Other triglycerides includeneutral oils, e.g., neutral plant oils, in particular fractionatedcoconut oils such as known and commercially available under the tradename MIGLYOL, including the products: MIGLYOL 810; MIGLYOL 812; MIGLYOL818; and CAPTEX 355. Other triglycerides are caprylic-capric acidtriglycerides such as known and commercially available under the tradename MYRITOL, including the product MYRITOL 813. Further triglyceridesof this class are CAPMUL MCT, CAPTEX 200, CAPTEX 300, CAPTEX 800, NEOBEEM5 and MAZOL 1400.

Pharmaceutical compositions comprising triglycerides may furthercomprise lipophilic and/or hydrophilic surfactants which may form clearsolutions upon dissolution with an aqueous solvent. One such surfactantis tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS).Examples of such compositions are described in U.S. Pat. No. 6,267,985.

In another embodiment, the pharmaceutically acceptable carrier comprisesLABRASOL (Gattefosse SA), which is PEG-8 caprylic/capric glycerides. Inanother embodiment, the pharmaceutically acceptable carrier comprisesPL90G, vitamin E TPGS, and Miglyol 812N. The components of such aformulation are shown in Table 1.

TABLE 1 Concentration (mg/ml) Ingredient Placebo 30 mg/ml3,5-Dimethyl-benzoic acid 0 mg 30 mg (R)—N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy- benzoyl)-hydrazide PL90G [Phospholipon 100 mg 100mg 90G] Vitamin E TPGS 100 mg 100 mg BHT [butylated 0.1 mg 0.1 mghydroxytoluene] Miglyol 812N [Medium QS to 1 ml QS to 1 ml chaintriglycerides] Li-caps [hard gelatine 1 per dose 1 per dose capsules]

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of one ormore of the ligands with a suppository base. Suitable suppository basesare, for example, natural or synthetic triglycerides, or paraffinhydrocarbons. In addition, it is also possible to use gelatin rectalcapsules which consist of a combination of the ligand with a base.Possible base materials include, for example, liquid triglycerides,polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the ligand in water-soluble form, for example,water-soluble salts and alkaline solutions. In addition, suspensions ofthe ligand as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides or polyethylene glycol-400.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers.

The topical compositions may be formulated as oils, creams, lotions,ointments and the like by choice of appropriate carriers. Suitablecarriers include vegetable or mineral oils, white petrolatum (white softparaffin), branched chain fats or oils, animal fats and high molecularweight alcohol (greater than C₁₂). Emulsifiers, stabilizers, humectantsand antioxidants may also be included as well as agents imparting coloror fragrance, if desired. Additionally, transdermal penetrationenhancers can be employed in these topical formulations. Examples ofsuch enhancers can be found in U.S. Pat. Nos. 3,989,816 and 4,444,762.

Creams may be formulated from a mixture of mineral oil, self-emulsifyingbeeswax and water in which ligand, dissolved in a small amount of an oilsuch as almond oil, is admixed. A typical example of such a cream is onewhich includes about 40 parts water, about 20 parts beeswax, about 40parts mineral oil and about 1 part almond oil.

Ointments may be formulated by mixing a suspension of the ligand in avegetable oil such as almond oil with warm soft paraffin and allowingthe mixture to cool. A typical example of such an ointment is one whichincludes about 30% almond oil and about 70% white soft paraffin byweight.

Lotions may be conveniently prepared by preparing a suspension of theligand in a suitable high molecular weight alcohol such as propyleneglycol or polyethylene glycol.

Examples of antioxidants which may be added to the pharmaceuticalcompositions include BHA and BHT.

In one embodiment, the pharmaceutical composition comprises 30 mg ligandper mL LABRASOL in a solid gelatin capsule. In another embodiment, thecapsule contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg ligand.

Pharmaceutical compositions may contain from 0.01% to 99% by weight ofthe ligand. Compositions may be either in single or multiple dose forms.The amount of ligand in any particular pharmaceutical composition willdepend upon the effective dose, that is, the dose required to elicit thedesired gene expression or suppression. In one embodiment, 0.1 to 7.5mg/kg is administered to the subject. In another embodiment, 0.1 to 3mg/kg is administered to the subject. In another embodiment, 0.1 to 3mg/kg is administered.

Suitable routes of administering the pharmaceutical compositions includeoral, rectal, topical (including dermal, buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal, intra-tumoral and epidural) and bynaso-gastric tube. It will be understood by those skilled in the artthat the preferred route of administration will depend upon thecondition being treated and may vary with factors such as the conditionof the recipient. The pharmaceutical compositions may be administeredone or more times daily. In one embodiment, the pharmaceuticalcomposition is administered daily beginning 24 hours prior to theadministration of cells containing the ecdysone receptor complex and DNAbinding sequence.

The diacylhydrazine ligands of Formula I and chiral diacylhydrazineligands of Formula II or III of the present invention described hereinmay also be administered in conjunction with other pharmaceuticallyactive compounds. It will be understood by those skilled in the art thatpharmaceutically active compounds to be used in combination with theligands described herein will be selected in order to avoid adverseeffects on the recipient or undesirable interactions between thecompounds. Examples of other pharmaceutically active compounds which maybe used in combination with the ligands include, for example, AIDSchemotherapeutic agents, amino acid derivatives, analgesics,anesthetics, anorectal products, antacids and antiflatulents,antibiotics, anticoagulants, antidotes, antifibrinolytic agents,antihistamines, anti-inflamatory agents, antineoplastics,antiparasitics, antiprotozoals, antipyretics, antiseptics,antispasmodics and anticholinergics, antivirals, appetite suppressants,arthritis medications, biological response modifiers, bone metabolismregulators, bowel evacuants, cardiovascular agents, central nervoussystem stimulants, cerebral metabolic enhancers, cerumenolytics,cholinesterase inhibitors, cold and cough preparations, colonystimulating factors, contraceptives, cytoprotective agents, dentalpreparations, deodorants, dermatologicals, detoxifying agents, diabetesagents, diagnostics, diarrhea medications, dopamine receptor agonists,electrolytes, enzymes and digestants, ergot preparations, fertilityagents, fiber supplements, antifungal agents, galactorrhea inhibitors,gastric acid secretion inhibitors, gastrointestinal prokinetic agents,gonadotropin inhibitors, hair growth stimulants, hematinics,hemorrheologic agents, hemostatics, histamine H₂ receptor antagonists,hormones, hyperglycemic agents, hypolipidemics, immunosuppressants,laxatives, leprostatics, leukapheresis adjuncts, lung surfactants,migraine preparations, mucolytics, muscle relaxant antagonists, musclerelaxants, narcotic antagonists, nasal sprays, nausea medicationsnucleoside analogues, nutritional supplements, osteoporosispreparations, oxytocics, parasympatholytics, parasympathomimetics,Parkinsonism drugs, Penicillin adjuvants, phospholipids, plateletinhibitors, porphyria agents, prostaglandin analogues, prostaglandins,proton pump inhibitors, pruritus medications psychotropics, quinolones,respiratory stimulants, saliva stimulants, salt substitutes, sclerosingagents, skin wound preparations, smoking cessation aids, sulfonamides,sympatholytics, thrombolytics, Tourette's syndrome agents, tremorpreparations, tuberculosis preparations, uricosuric agents, urinarytract agents, uterine contractants, uterine relaxants, vaginalpreparations, vertigo agents, vitamin D analogs, vitamins, and medicalimaging contrast media. In some cases the ligands may be useful as anadjunct to drug therapy, for example, to “turn off” a gene that producesan enzyme that metabolizes a particular drug.

For agricultural applications, in addition to the applications describedabove, the ligands of this invention may also be used to control theexpression of pesticidal proteins such as Bacillus thuringiensis (Bt)toxin. Such expression may be tissue or plant specific. In addition,particularly when control of plant pests is also needed, one or morepesticides may be combined with the ligands described herein, therebyproviding additional advantages and effectiveness, including fewer totalapplications, than if the pesticides are applied separately. Whenmixtures with pesticides are employed, the relative proportions of eachcomponent in the composition will depend upon the relative efficacy andthe desired application rate of each pesticide with respect to thecrops, pests, and/or weeds to be treated. Those skilled in the art willrecognize that mixtures of pesticides may provide advantages such as abroader spectrum of activity than one pesticide used alone. Examples ofpesticides which can be combined in compositions with the ligandsdescribed herein include fungicides, herbicides, insecticides,miticides, and microbicides.

The diacylhydrazine ligands of Formula I and chiral diacylhydrazineligands of Formula II or III of the present invention described hereincan be applied to plant foliage as aqueous sprays by methods commonlyemployed, such as conventional high-liter hydraulic sprays, low-litersprays, air-blast, and aerial sprays. The dilution and rate ofapplication will depend upon the type of equipment employed, the methodand frequency of application desired, and the ligand application rate.It may be desirable to include additional adjuvants in the spray tank.Such adjuvants include surfactants, dispersants, spreaders, stickers,antifoam agents, emulsifiers, and other similar materials described inMcCutcheon's Emulsifiers and Detergents, McCutcheon's Emulsifiers andDetergents/Functional Materials, and McCutcheon's Functional Materials,all published annually by McCutcheon Division of MC Publishing Company(New Jersey). These ligands can also be mixed with fertilizers orfertilizing materials before their application. These ligands and solidfertilizing material can also be admixed in mixing or blendingequipment, or they can be incorporated with fertilizers in granularformulations. Any relative proportion of fertilizer can be used which issuitable for the crops and weeds to be treated. The ligands describedherein will commonly comprise from 5% to 50% of the fertilizingcomposition. These compositions provide fertilizing materials whichpromote the rapid growth of desired plants, and at the same time controlgene expression.

Host Cells and Non-Human Organisms

As described above, diacylhydrazine ligands of Formula I and chiraldiacylhydrazine ligands of Formula II or III may be used to modulategene expression in a host cell. Expression in transgenic host cells maybe useful for the expression of various genes of interest. The presentinvention provides ligands for modulation of gene expression inprokaryotic and eukaryotic host cells.

Expression in transgenic host cells is useful for the expression ofvarious polypeptides of interest including, but not limited to, antigensproduced in plants as vaccines; enzymes like alpha-amylase, phytase,glucanes, and xylanase; genes for resistance against insects, nematodes,fungi, bacteria, viruses, and abiotic stresses in plants; antigens;nutraceuticals; pharmaceuticals; vitamins; genes for modifying aminoacid content, herbicide resistance, cold, drought, and heat tolerance;industrial products; oils, protein, carbohydrates; antioxidants; malesterile plants; flowers; fuels; other output traits; therapeuticpolypeptides or products that may be used to treat a condition, adisease, a disorder, a dysfunction, or a genetic defect, such asmonoclonal antibodies, enzymes, proteases, cytokines, interferons,insulin, erythropoietin, clotting factors, other blood factors orcomponents; viral vectors for gene therapy; virus for vaccines; targetsfor drug discovery, functional genomics, and proteomics analyses andapplications: pathway intermediates for the modulation of pathwaysalready existing in the host; pathway intermediates for the synthesis ofnew products heretofore not possible using the host; cell based assays;functional genomics assays; proteomics assays, and the like.Additionally the gene products may be useful for conferring highergrowth yields of the host or for enabling an alternative growth mode tobe utilized.

Thus, the present invention provides diacylhydrazine ligands of FormulaI and chiral diacylhydrazine ligands of Formula II or III for modulatinggene expression in an isolated host cell. In one embodiment, theisolated host cell is a prokaryotic host cell or a eukaryotic host cell.In another embodiment, the isolated host cell is an invertebrate hostcell or a vertebrate host cell. Preferably, the host cell is selectedfrom the group consisting of a bacterial cell, a fungal cell, a yeastcell, a nematode cell, an insect cell, a fish cell, a plant cell, anavian cell, an animal cell, and a mammalian cell. More preferably, thehost cell is a yeast cell, a nematode cell, an insect cell, a plantcell, a zebrafish cell, a chicken cell, a hamster cell, a mouse cell, arat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goatcell, a cow cell, a pig cell, a horse cell, a sheep cell, a simian cell,a monkey cell, a chimpanzee cell, or a human cell. Examples of hostcells include, but are not limited to, fungal or yeast species such asAspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, orbacterial species such as those in the genera Synechocystis,Synechococcus, Salmonella, Bacillus, Acinetobacter, Rhodococcus,Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter,Alcaligenes, Anabaena, Thiobacillus, Methanobacterium and Klebsiella;plant species selected from the group consisting of apple, Arabidopsis,bajra, banana, barley, beans, beet, blackgram, chickpea, chili,cucumber, eggplant, favabean, maize, melon, millet, mungbean, oat, okra,Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus,potato, pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet,sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat;animal; and mammalian host cells.

In another embodiment, the host cell is a yeast cell selected from thegroup consisting of a Saccharomyces, a Pichia, and a Candida host cell.

In another embodiment, the host cell is a Caenorhabditis elegansnematode cell.

In another embodiment, the host cell is an insect cell.

In another embodiment, the host cell is a plant cell selected from thegroup consisting of an apple, Arabidopsis, bajra, banana, barley, beans,beet, blackgrum, chickpea, chili, cucumber, eggplant, favabean, maize,melon, millet, mungbean, oat, okra, Panicum, papaya, peanut, pea,pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin, rice, sorghum,soybean, squash, sugarcane, sugarbeet, sunflower, sweet potato, tea,tomato, tobacco, watermelon, and wheat cell.

In another embodiment, the host cell is a zebrafish cell.

In another embodiment, the host cell is a chicken cell.

In another embodiment, the host cell is a mammalian cell selected fromthe group consisting of a hamster cell, a mouse cell, a rat cell, arabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell, a cowcell, a pig cell, a horse cell, a sheep cell, a monkey cell, achimpanzee cell, and a human cell.

Host cell transformation is well known in the art and may be achieved bya variety of methods including but not limited to electroporation, viralinfection, plasmid/vector transfection, non-viral vector mediatedtransfection, Agrobacterium-mediated transformation, particlebombardment, and the like. Expression of desired gene products involvesculturing the transformed host cells under suitable conditions andinducing expression of the transformed gene. Culture conditions and geneexpression protocols in prokaryotic and eukaryotic cells are well knownin the art. Cells may be harvested and the gene products isolatedaccording to protocols specific for the gene product.

In addition, a host cell may be chosen which modulates the expression ofthe inserted polynucleotide, or modifies and processes the polypeptideproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g., glycosylation,cleavage (e.g., of signal sequence)) of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce a non-glycosylated coreprotein product. However, a polypeptide expressed in bacteria may not beproperly folded. Expression in yeast can produce a glycosylated product.Expression in eukaryotic cells can increase the likelihood of “native”glycosylation and folding of a heterologous protein. Moreover,expression in mammalian cells can provide a tool for reconstituting, orconstituting, the polypeptide's activity. Furthermore, differentvector/host expression systems may affect processing reactions, such asproteolytic cleavages, to a different extent. The diacylhydrazineligands of Formula I and chiral diacylhydrazine ligands of Formula II orIII of the present invention may be used in a non-human organismcomprising an isolated host cell. In one embodiment, the non-humanorganism is a prokaryotic organism or a eukaryotic organism. In anotherembodiment, the non-human organism is an invertebrate organism or avertebrate organism.

Preferably, the non-human organism is selected from the group consistingof a bacterium, a fungus, a yeast, a nematode, an insect, a fish, aplant, a bird, an animal, and a mammal. More preferably, the non-humanorganism is a yeast, a nematode, an insect, a plant, a zebrafish, achicken, a hamster, a mouse, a rat, a rabbit, a cat, a dog, a bovine, agoat, a cow, a pig, a horse, a sheep, a simian, a monkey, or achimpanzee.

In another embodiment, the non-human organism is a yeast selected fromthe group consisting of Saccharomyces, Pichia, and Candida.

In another embodiment, the non-human organism is a Caenorhabditiselegans nematode.

In another embodiment, the non-human organism is a plant selected fromthe group consisting of apple, Arabidopsis, bajra, banana, barley,beans, beet, blackgum, chickpea, chili, cucumber, eggplant, favabean,maize, melon, millet, mungbean, oat, okra, Panicum, papaya, peanut, pea,pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin, rice, sorghum,soybean, squash, sugarcane, sugarbeet, sunflower, sweet potato, tea,tomato, tobacco, watermelon, and wheat.

In another embodiment, the non-human organism is a Mus musculus mouse.

Gene Expression Modulation Systems

The present invention pertains to diacylhydrazine ligands of Formula Iand chiral diacylhydrazine ligands of Formula II or III that are usefulin an ecdysone receptor-based inducible gene expression system. Thesediacylhydrazine ligands provide an improved inducible gene expressionsystem in both prokaryotic and eukaryotic host cells. Thus, the presentinvention pertains to diacylhydrazine ligands of Formula I and chiraldiacylhydrazine ligands of Formula II or III that are useful to modulateexpression of genes. In particular, the present invention pertains todiacylhydrazine ligands of Formula I and chiral diacylhydrazine ligandsof Formula II or III having the ability to transactivate a geneexpression modulation system comprising at least one gene expressioncassette that is capable of being expressed in a host cell comprising apolynucleotide that encodes a polypeptide comprising a Group H nuclearreceptor ligand binding domain. In one embodiment, the Group H nuclearreceptor ligand binding is from an ecdysone receptor, a ubiquitousreceptor, an orphan receptor 1, a NER-1, a steroid hormone nuclearreceptor 1, a retinoid X receptor interacting protein-15, a liver Xreceptor β, a steroid hormone receptor like protein, a liver X receptor,a liver X receptor α, a farnesoid X receptor, a receptor interactingprotein 14, or a farnesol receptor. In another embodiment, the Group Hnuclear receptor ligand binding domain is from an ecdysone receptor.

In another embodiment, the gene expression modulation system comprises agene expression cassette comprising a polynucleotide that encodes apolypeptide comprising a transactivation domain, a DNA-binding domainthat recognizes a response element associated with a gene whoseexpression is to be modulated; and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation. The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another embodiment, the gene expression modulation system comprises agene expression cassette comprising a) a polynucleotide that encodes apolypeptide comprising a transactivation domain, a DNA-binding domainthat recognizes a response element associated with a gene whoseexpression is to be modulated; and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, and b) a secondnuclear receptor ligand binding domain selected from the groupconsisting of a vertebrate retinoid X receptor ligand binding domain, aninvertebrate retinoid X receptor ligand binding domain, an ultraspiracleprotein ligand binding domain, and a chimeric ligand binding domaincomprising two polypeptide fragments, wherein the first polypeptidefragment is from a vertebrate retinoid X receptor ligand binding domain,an invertebrate retinoid X receptor ligand binding domain, or anultraspiracle protein ligand binding domain, and the second polypeptidefragment is from a different vertebrate retinoid X receptor ligandbinding domain, invertebrate retinoid X receptor ligand binding domain,or ultraspiracle protein ligand binding domain. The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another embodiment, the gene expression modulation system comprises afirst gene expression cassette comprising a polynucleotide that encodesa first polypeptide comprising a DNA-binding domain that recognizes aresponse element associated with a gene whose expression is to bemodulated and a nuclear receptor ligand binding domain, and a secondgene expression cassette comprising a polynucleotide that encodes asecond polypeptide comprising a transactivation domain and a nuclearreceptor ligand binding domain, wherein one of the nuclear receptorligand binding domains is a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation. In one embodiment, the firstpolypeptide is substantially free of a transactivation domain and thesecond polypeptide is substantially free of a DNA binding domain. Forpurposes of the invention, “substantially free” means that the proteinin question does not contain a sufficient sequence of the domain inquestion to provide activation or binding activity. The gene expressionmodulation system may further comprise a third gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the first polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the secondpolypeptide of the second gene expression cassette; and iii) a genewhose expression is to be modulated.

Wherein when only one nuclear receptor ligand binding domain is a GroupH ligand binding domain comprising a substitution mutation, the othernuclear receptor ligand binding domain may be from any other nuclearreceptor that forms a dimer with the Group H ligand binding domaincomprising the substitution mutation. For example, when the Group Hnuclear receptor ligand binding domain comprising a substitutionmutation is an ecdysone receptor ligand binding domain comprising asubstitution mutation, the other nuclear receptor ligand binding domain(“partner”) may be from an ecdysone receptor, a vertebrate retinoid Xreceptor (RXR), an invertebrate RXR, an ultraspiracle protein (USP), ora chimeric nuclear receptor comprising at least two different nuclearreceptor ligand binding domain polypeptide fragments selected from thegroup consisting of a vertebrate RXR, an invertebrate RXR, and a USP(see WO 01/70816 A2, International Patent Application No. PCT/US02/05235and US 2004/0096942 A1, incorporated herein by reference in theirentirety). The “partner” nuclear receptor ligand binding domain mayfurther comprise a truncation mutation, a deletion mutation, asubstitution mutation, or another modification.

In one embodiment, the vertebrate RXR ligand binding domain is from ahuman Homo sapiens, mouse Mus musculus, rat Rattus norvegicus, chickenGallus gallus, pig Sus scrofa domestica, frog Xenopus laevis, zebrafishDanio rerio, tunicate Polyandrocarpa misakiensis, or jellyfishTripedalia cysophora RXR.

In one embodiment, the invertebrate RXR ligand binding domain is from alocust Locusta migratoria ultraspiracle polypeptide (“LmUSP”), an ixodidtick Amblyomma americanum RXR homolog 1 (“AmaRXR1”), an ixodid tickAmblyomma americanum RXR homolog 2 (“AmaRXR2”), a fiddler crab Celucapugilator RXR homolog (“CpRXR”), a beetle Tenebrio molitor RXR homolog(“TmRXR”), a honeybee Apis mellifera RXR homolog (“AmRXR”), an aphidMyzus persicae RXR homolog (“MpRXR”), or a non-Dipteran/non-LepidopteranRXR homolog.

In one embodiment, the chimeric RXR ligand binding domain comprises atleast two polypeptide fragments selected from the group consisting of avertebrate species RXR polypeptide fragment, an invertebrate species RXRpolypeptide fragment, and a non-Dipteran/non-Lepidopteran invertebratespecies RXR homolog polypeptide fragment. A chimeric RXR ligand bindingdomain for use in the present invention may comprise at least twodifferent species RXR polypeptide fragments, or when the species is thesame, the two or more polypeptide fragments may be from two or moredifferent isoforms of the species RXR polypeptide fragment.

In one embodiment, the chimeric RXR ligand binding domain comprises atleast one vertebrate species RXR polypeptide fragment and oneinvertebrate species RXR polypeptide fragment.

In another embodiment, the chimeric RXR ligand binding domain comprisesat least one vertebrate species RXR polypeptide fragment and onenon-Dipteran/non-Lepidopteran invertebrate species RXR homologpolypeptide fragment.

In another embodiment, the gene whose expression is to be modulated is ahomologous gene with respect to the host cell. In another embodiment,the gene whose expression is to be modulated is a heterologous gene withrespect to the host cell.

The diacylhydrazine ligands of Formula I and chiral diacylhydrazineligands of Formula II or III for use in the present invention asdescribed below, when combined with the ligand binding domain of thenuclear receptor(s), which in turn are bound to the response elementlinked to a gene, provide the means for external temporal regulation ofexpression of the gene. The binding mechanism or the order in which thevarious components of this invention bind to each other, that is, forexample, ligand to ligand binding domain, DNA-binding domain to responseelement, transactivation domain to promoter, etc., is not critical.

The ecdysone receptor is a member of the nuclear receptor superfamilyand classified into subfamily 1, group H (referred to herein as “Group Hnuclear receptors”). The members of each group share 40-60% amino acididentity in the E (ligand binding) domain (Laudet et al., Cell97:161-163 (1999)). In addition to the ecdysone receptor, other membersof this nuclear receptor subfamily 1, group H include: ubiquitousreceptor (UR), orphan receptor 1 (OR-1), steroid hormone nuclearreceptor 1 (NER-1), retinoid X receptor interacting protein-15 (RIP-15),liver X receptor β (LXRβ), steroid hormone receptor like protein(RLD-1), liver X receptor (LXR), liver X receptor α (LXRα), farnesoid Xreceptor (FXR), receptor interacting protein 14 (RIP-14), and farnesolreceptor (HRR-1).

In a specific example, binding of the ligand to the ligand bindingdomain of a Group H nuclear receptor and its nuclear receptor ligandbinding domain partner enables expression or suppression of the gene.This mechanism does not exclude the potential for ligand binding to theGroup H nuclear receptor (GHNR) or its partner, and the resultingformation of active homodimer complexes (e.g. GHNR+GHNR orpartner+partner). Preferably, one or more of the receptor domains isvaried producing a hybrid gene switch. Typically, one or more of thethree domains, DBD, LBD, and transactivation domain, may be chosen froma source different than the source of the other domains so that thehybrid genes and the resulting hybrid proteins are optimized in thechosen host cell or organism for transactivating activity, complementarybinding of the ligand, and recognition of a specific response element.In addition, the response element itself can be modified or substitutedwith response elements for other DNA binding protein domains such as theGAL-4 protein from yeast (see Sadowski et al., Nature 335:563-564(1988)) or LexA protein from Escherichia coli (see Brent et al., Cell43:729-736 (1985)), or synthetic response elements specific for targetedinteractions with proteins designed, modified, and selected for suchspecific interactions (see, for example, Kim et al., Proc. Natl. Acad.Sci. USA, 94:3616-3620 (1997)) to accommodate hybrid receptors. Anotheradvantage of two-hybrid systems is that they allow choice of a promoterused to drive the gene expression according to a desired end result.Such double control can be particularly important in areas of genetherapy, especially when cytotoxic proteins are produced, because boththe timing of expression as well as the cells wherein expression occurscan be controlled. When genes, operably linked to a suitable promoter,are introduced into the cells of the subject, expression of theexogenous genes is controlled by the presence of the system of thisinvention. Promoters may be constitutively or inducibly regulated or maybe tissue-specific (that is, expressed only in a particular type ofcells) or specific to certain developmental stages of the organism.

In particular, described herein are diacylhydrazine ligands of Formula Iand chiral diacylhydrazine ligands of Formula II or III useful in a geneexpression modulation system comprising a Group H nuclear receptorligand binding domain comprising a substitution mutation. This geneexpression system may be a “single switch”-based gene expression systemin which the transactivation domain, DNA-binding domain and ligandbinding domain are on one encoded polypeptide. Alternatively, the geneexpression modulation system may be a “dual switch”- or“two-hybrid”-based gene expression modulation system in which thetransactivation domain and DNA-binding domain are located on twodifferent encoded polypeptides.

An ecdysone receptor-based gene expression modulation system for use inthe present invention may be either heterodimeric or homodimeric. Afunctional EcR complex generally refers to a heterodimeric proteincomplex consisting of two members of the steroid receptor family, anecdysone receptor protein obtained from various insects, and anultraspiracle (USP) protein or the vertebrate homolog of USP, retinoid Xreceptor protein (see Yao et al., Nature 366:476-479 (1993) and Yao etal., Cell 71:63-72 (1992)). However, the complex may also be a homodimeras detailed below. The functional ecdysteroid receptor complex may alsoinclude additional protein(s) such as immunophilins. Additional membersof the steroid receptor family of proteins, known as transcriptionalfactors (such as DHR38 or betaFTZ-1), may also be ligand dependent orindependent partners for EcR, USP, and/or RXR. Additionally, othercofactors may be required such as proteins generally known ascoactivators (also termed adapters or mediators). These proteins do notbind sequence-specifically to DNA and are not involved in basaltranscription. They may exert their effect on transcription activationthrough various mechanisms, including stimulation of DNA-binding ofactivators, by affecting chromatin structure, or by mediatingactivator-initiation complex interactions. Examples of such coactivatorsinclude RIP140, TIF1, RAP46/Bag-1, ARA70, SRC-1/NCoA-1,TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the promiscuouscoactivator C response element B binding protein, CBP/p300 (for reviewsee Glass et al., Curr. Opin. Cell Biol. 9:222-232 (1997)). Also,protein cofactors generally known as corepressors (also known asrepressors, silencers, or silencing mediators) may be required toeffectively inhibit transcriptional activation in the absence of ligand.These corepressors may interact with the unliganded ecdysone receptor tosilence the activity at the response element. Current evidence suggeststhat the binding of ligand changes the conformation of the receptor,which results in release of the corepressor and recruitment of the abovedescribed coactivators, thereby abolishing their silencing activity.Examples of corepressors include N—CoR and SMRT (for review, see Horwitzet al., Mol. Endocrinol. 10: 1167-1177 (1996)). These cofactors mayeither be endogenous within the cell or organism, or may beadded-exogenously as transgenes to be expressed in either a regulated orunregulated fashion. Homodimer complexes of the ecdysone receptorprotein, USP, or RXR may also be functional under some circumstances.

The ecdysone receptor complex typically includes proteins that aremembers of the nuclear receptor superfamily wherein all members aregenerally characterized by the presence of an amino-terminaltransactivation domain, a DNA binding domain (“DBD”), and a ligandbinding domain (“LBD”) separated from the DBD by a hinge region. As usedherein, the term “DNA binding domain” comprises a minimal polypeptidesequence of a DNA binding protein, up to the entire length of a DNAbinding protein, so long as the DNA binding domain functions toassociate with a particular response element. Members of the nuclearreceptor superfamily are also characterized by the presence of four orfive domains: A/B, C, D, E, and in some members F (see U.S. Pat. No.4,981,784 and Evans, Science 240:889-895 (1988)). The “A/B” domaincorresponds to the transactivation domain, “C” corresponds to the DNAbinding domain, “D” corresponds to the hinge region, and “E” correspondsto the ligand binding domain. Some members of the family may also haveanother transactivation domain on the carboxy-terminal side of the LBDcorresponding to “F”.

The DBD is characterized by the presence of two cysteine zinc fingersbetween which are two amino acid motifs, the P-box and the D-box, whichconfer specificity for ecdysone response elements. These domains may beeither native, modified, or chimeras of different domains ofheterologous receptor proteins. The EcR receptor, like a subset of thesteroid receptor family, also possesses less well-defined regionsresponsible for heterodimerization properties. Because the domains ofnuclear receptors are modular in nature, the LBD, DBD, andtransactivation domains may be interchanged.

Gene switch systems are known that incorporate components from theecdysone receptor complex. However, in these known systems, whenever EcRis used it is associated with native or modified DNA binding domains andtransactivation domains on the same molecule. USP or RXR are typicallyused as silent partners. It has previously been shown that when DNAbinding domains and transactivation domains are on the same molecule thebackground activity in the absence of ligand is high and that suchactivity is dramatically reduced when DNA binding domains andtransactivation domains are on different molecules, that is, on each oftwo partners of a heterodimeric or homodimeric complex (seePCT/US01/09050).

Method of Modulating Gene Expression

The present invention also relates to methods of modulating geneexpression in a host cell using a gene expression modulation system andthe ligands of the present invention. Specifically, the presentinvention provides a method of modulating the expression of a gene in ahost cell comprising the steps of: a) introducing into the host cell agene expression modulation system; and b) introducing into the host cella diacylhydrazine ligand of Formula I or chiral diacylhydrazine ligandof Formula II or III; wherein the gene to be modulated is a component ofa gene expression cassette comprising: i) a response element comprisinga domain recognized by the DNA binding domain of the gene expressionsystem; ii) a promoter that is activated by the transactivation domainof the gene expression system; and iii) a gene whose expression is to bemodulated, whereby upon introduction of the diacylhydrazine ligand ofFormula I or chiral diacylhydrazine ligand of Formula II or III into thehost cell, expression of the gene is modulated.

The present invention also provides a method of modulating theexpression of a gene in a host cell comprising the steps of: a)introducing into the host cell a gene expression modulation system; b)introducing into the host cell a gene expression cassette, wherein thegene expression cassette comprises i) a response element comprising adomain recognized by the DNA binding domain from the gene expressionsystem; ii) a promoter that is activated by the transactivation domainof the gene expression system; and iii) a gene whose expression is to bemodulated; and c) introducing into the host cell a diacylhydrazineligand of Formula I or chiral diacylhydrazine ligand of Formula II orIII; whereby upon introduction of the diacylhydrazine ligand of FormulaI or chiral diacylhydrazine ligand of Formula II or III into the hostcell, expression of the gene is modulated.

The present invention also provides a method of modulating theexpression of a gene in a host cell comprising a gene expressioncassette comprising a response element comprising a domain to which theDNA binding domain from the first hybrid polypeptide of the geneexpression modulation system binds; a promoter that is activated by thetransactivation domain of the second hybrid polypeptide of the geneexpression modulation system; and a gene whose expression is to bemodulated; wherein the method comprises the steps of: a) introducinginto the host cell a gene expression modulation system; and b)introducing into the host cell a diacylhydrazine ligand of Formula I orchiral diacylhydrazine ligand of Formula II or III; whereby uponintroduction of the ligand into the host, expression of the gene ismodulated.

Genes of interest for expression in a host cell using methods disclosedherein may be endogenous genes or heterologous genes. Nucleic acid oramino acid sequence information for a desired gene or protein can belocated in one of many public access databases, for example, GENBANK,EMBL, Swiss-Prot, and PIR, or in many biology related journalpublications. Thus, those skilled in the art have access to nucleic acidsequence information for virtually all known genes. Such information canthen be used to construct the desired constructs for the insertion ofthe gene of interest within the gene expression cassettes used in themethods described herein.

Measuring Gene Expression/Transcription

One useful measurement of the methods of the invention is that of thetranscriptional state of the cell including the identities andabundances of RNA, preferably mRNA species. Such measurements areconveniently conducted by measuring cDNA abundances by any of severalexisting gene expression technologies.

Nucleic acid array technology is a useful technique for determiningdifferential mRNA expression. Such technology includes, for example,oligonucleotide chips and DNA microarrays. These techniques rely on DNAfragments or oligonucleotides which correspond to different genes orcDNAs which are immobilized on a solid support and hybridized to probesprepared from total mRNA pools extracted from cells, tissues, or wholeorganisms and converted to cDNA. Oligonucleotide chips are arrays ofoligonucleotides synthesized on a substrate using photolithographictechniques. Chips have been produced which can be used for analysis ofup to 1700 genes. DNA microarrays are arrays of DNA samples, typicallyPCR products, that are robotically printed onto a microscope slide. Eachgene is analyzed by a full or partial-length target DNA sequence.Microarrays with up to 10,000 genes are now routinely preparedcommercially. The primary difference between these two techniques isthat oligonucleotide chips typically utilize 25-mer oligonucleotideswhich allow fractionation of short DNA molecules whereas the larger DNAtargets of microarrays, approximately 1000 base pairs, may provide moresensitivity in fractionating complex DNA mixtures.

Another useful measurement of the methods of the invention is that ofdetermining the translation state of the cell by measuring theabundances of the constituent protein species present in the cell usingprocesses well known in the art.

Where identification of genes associated with various physiologicalfunctions is desired, an assay may be employed in which changes in suchfunctions as cell growth, apoptosis, senescence, differentiation,adhesion, binding to a specific molecules, binding to another cell,cellular organization, organogenesis, intracellular transport, transportfacilitation, energy conversion, metabolism, myogenesis, neurogenesis,and/or hematopoiesis is measured.

In addition, selectable marker or reporter gene expression may be usedto measure gene expression modulation using the present invention.

Other methods to detect the products of gene expression are well knownin the art and include Southern blots (DNA detection), dot or slot blots(DNA, RNA), northern blots (RNA), RT-PCR (RNA), western blots(polypeptide detection), and ELISA (polypeptide) analyses. Although lesspreferred, labeled proteins can be used to detect a particular nucleicacid sequence to which it hybridizes.

In some cases it is necessary to amplify the amount of a nucleic acidsequence. This may be carried out using one or more of a number ofsuitable methods including, for example, polymerase chain reaction(“PCR”), ligase chain reaction (“LCR”), strand displacementamplification (“SDA”), transcription-based amplification, and the like.PCR is carried out in accordance with known techniques in which, forexample, a nucleic acid sample is treated in the presence of a heatstable DNA polymerase, under hybridizing conditions, with one pair ofoligonucleotide primers, with one primer hybridizing to one strand(template) of the specific sequence to be detected. The primers aresufficiently complementary to each template strand of the specificsequence to hybridize therewith. An extension product of each primer issynthesized and is complementary to the nucleic acid template strand towhich it hybridized. The extension product synthesized from each primercan also serve as a template for further synthesis of extension productsusing the same primers. Following a sufficient number of rounds ofsynthesis of extension products, the sample may be analyzed as describedabove to assess whether the sequence or sequences to be detected arepresent.

General Methods

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) (Maniatis) andby T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984)and by Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Assoc. and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials used for the growth and maintenance of host cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma ChemicalCompany (St. Louis, Mo.) unless otherwise specified.

Manipulations of genetic sequences may be accomplished using the suiteof programs available from the Genetics Computer Group Inc. (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.).Where the GCG program “Pileup” is used the gap creation default value of12, and the gap extension default value of 4 may be used. Where the CGC“Gap” or “Bestfit” program is used the default gap creation penalty of50 and the default gap extension penalty of 3 may be used. In any casewhere GCG program parameters are not prompted for, in these or any otherGCG program, default values may be used.

Melting points were measured in glass capillary tubes with anElectrothermal® apparatus and are uncorrected. Optical rotations ([α]²⁵₅₈₉) were measured at room temperature in a 10 cm long quartz cell usinga Perkin Elmer Model 341 Polarimeter. Concentrations are given in g/100mL. ¹H NMR spectra were recorded at 300 MHz or 400.13 MHz using a BrukerNMR. Unless otherwise stated, the internal reference was solvent. ¹³CNMR spectra were recorded at 100.6 MHz with a Bruker NMR. Unlessotherwise stated, the internal reference was solvent. LC-MS analysis wasperformed using an Agilent 1100 LC stack coupled with an Agilent singlequad mass spectrometer. Solvents were (A) H₂O/0.1% formic acid and (B)ACN/0.1% formic acid in a gradient of T=0 15% B to T=10 98% B and a stoptime of 20 min on a 75 mm×2.1 mm C18 column with a flow rate=0.2 mL/min.Exact mass analysis were performed by direct infusion into an AgilentESI/TOF mass spectrometer. X-Ray crystal structure determinations wereperformed using a Bruker SMART6000. Elemental analyses were performed ona Perkin Elmer 2400 CHN analyzer. Analytical thin layer chromatographyor TLC was performed using Macherey-Nagel Polygram® Sil G/UV₂₅₄ 0.2 mmplates. Most plates were visualized by UV light. Some were developedusing iodine or phosphomolybdic acid. Silica gel chromatography wasperformed using Aldrich silica gel (230-400 mesh, 60 Å) in glass columnsunder a N₂ or argon head pressure of ca. 30 psi. Analytical HPLC wasperformed using a Gilson HPLC system equipped with a 811C dynamic mixer,306 UV/VIS 155 detector, and 215 liquid handler. UV absorbance wasmeasured at 220 nm and 254 nm and integrations were performed at 254 nm.Flow rates were typically held at 1 mL/min. Normal phase chromatographywas performed with a 4.6 mm×25 cm DuPont Zorbax ODS column. Reversephase chromatography was performed using an Alltech Adsorbosphere 5micron, 4.6 mm×25 cm C18 column. Chiral HPLC was performed using aCHIRALCEL® 5 micron, 4.6 mm×25 cm OD-H column, a CHIRAKPAK® 5 micron,4.6 mm×25 cm AD-H column, or a 5 micron, 100 Å, 4.6 mm×25 cm RegisRexchrom (S,S) ULMO column. Solvents were reagent grade unless otherwisestated. Anhydrous solvents were used as purchased.

General Synthetic Methods

Enantiomerically enriched compounds of Formula II or III where A, B andR² are defined as above and R¹ is alkenyl may be prepared via asymmetricsynthesis as described in General Scheme 1.

Reaction of protected hydrazine 1 (e.g.; benzyl carbazate or t-butylcarbazate) with aldehyde R²CHO affords 2. Asymmetric allylation of 2provides either 3 or 4 in enantiomerically enriched form, dependinginter alia on the choice of chiral reagent and R². Methods to performasymmetric allylation reactions are known in the art and may be utilizedto prepare compounds of the invention. The synthesis of chiral reagentsparticularly useful in asymmetric allylation reactions is described inLeighton et al., J. Am. Chem. Soc. 125:9596 (2003) and WO 03/074534.Reaction of 3 with carboxylic acid chloride B—COCl gives 5. Carboxylicacid B—CO₂H may also be coupled with 3 to provide 5. Removal of theprotecting group of 5 gives 6. One skilled in the art recognizes thereare a variety of methods to deprotect a hydrazine, depending on thenature of the protecting group. Suitable protection/deprotectionstrategies are discussed in “Protective Groups in Organic Synthesis”, T.W. Green and P. G. M. Wuts (1999). Reaction of 7 with carboxylic acidchloride A-COCl or carboxylic acid A-CO₂H gives 8, a compound of FormulaII. Similarly, reaction of 4 leads to a compound of Formula III.

Enantiomerically enriched compounds of Formula II or III where A, B, R¹and R² are defined as above may also be prepared via asymmetricreduction as described in General Scheme 2.

Reaction of acyl hydrazine 1 with ketone R¹COR² provides hydrazone 2.Asymmetric reduction of 2 provides 3 or 4 in enantiomerically enrichedform, depending inter alia on the choice of reducing agent, R¹ and R².U.S. Pat. No. 5,250,731 describes the asymmetric catalytic hydrogenationof acyl hydrazones to give optically active acyl hydrazines in thepresence of a chiral phospholane catalyst complex. One skilled in theart will recognize other chiral ligands may also be used. Moreover, oneskilled in the art will recognize additional asymmetric reductionmethods may employed. In this example, reaction of 3 with BCOCl gives 5,a compound of Formula II. Similarly, reaction of 4 leads to a compoundof Formula III.

In addition, enantiomerically enriched compounds of Formula II or IIImay be prepared via chiral resolution, or a combination of asymmetricsynthesis and chiral resolution or a combination of asymmetrichydrogenation and chiral resolution. As described in General Scheme 3,subjecting racemic diacylhydrazines to chiral HPLC chromatographyprovides enantiomerically enriched compounds of Formula II or III.Suitable chiral columns for use in chiral resolutions include, forexample, Daicel CHIRALCEL® OD-H, Daicel CHIRAKPAK® AD-H and RegisTechnologies ULMO chiral columns. Other chiral resolution methods arealso possible. Racemic diacylhydrazines for use in chiral resolutionprocesses may be prepared using methodology described in US 2005/0209283A1 and US 2006/0020146 A1.

Enantiomerically enriched compounds of Formula II or III where R¹ isalkenyl may be further elaborated using standard chemicaltransformations. As described in General Scheme 4 for anenantiomerically enriched compound of Formula III, the alkenyl may beconverted to an alkyl, a hydroxyalkyl, an alkyl optionally substitutedwith a cycloalkyl, an alkyl optionally substituted with a heterocycle,an alkyl substituted with an alkoxy, a haloalkyl and other optionallysubstituted alkyl groups. One skilled in the art will recognize anassortment of single or multi-step chemical transformations that may beused to convert an alkene to other groups of the invention.

EXAMPLES

The present invention may be better understood by reference to thefollowing general synthetic methods provided above and non-limitingexamples provided below, which are provided as exemplary of theinvention.

Example 1

Synthesis

Compound 1: Benzyl carbazate is commercially available.

Compound 2: Benzyl carbazate (300 g, 1.81 moles) was dissolved in 1.2 Lof methyl alcohol in a 3 L 4-neck flask equipped with reflux condenser,thermometer, magnetic stirring, and a 500 mL addition funnel. Glacialacetic acid (5 mL) was added to the mixture that was then brought to 45°C. Pivaldehyde (248.8 g of solution, 2.17 moles, ca. 75% in t-butanol)was added portionwise over 30 minutes. The reaction was heated at refluxfor 30 minutes, while monitoring by TLC. The mixture was allowed to coolto nearly room temperature and was concentrated on a rotary evaporatorto a total volume of ca. 700 mL. Ca. 7 g activated charcoal was added atca. 30° C. The mixture was stirred overnight and filtered through a padof Celite in a pore size “C” glass fritted Büchner funnel. The solventwas removed in vacuo to leave a light yellow oil that was poured into acrystallizing dish and induced to crystallize by manipulation with aspatula. The material was granulated and residual solvent was allowed toevaporate in air at room temperature for several days, leaving 420.4 gcrude product. Recrystallization at room temperature from an initiallyboiling mixture of 300 mL ethyl acetate and 1 L hexanes produced 363.2 gof (E)-N′-(2,2-dimethyl-propylidene)-hydrazinecarboxylic acid benzylester as an off-white solid. A second crop of 43.1 g and identicalmelting point was obtained from a mixture of ca. 150 mL hexanes and 45mL ethyl acetate. Total yield: 96.1%. R_(f)=0.32 (2:1 hexanes:ethylacetate); mp=74-74.5° C.; ¹H NMR (400 MHz, CDCl₃) δ 8.0 (1H, br s),7.4-7.3 (5H, m), 7.1 (1H, s), 5.20 (2H, s), 1.1 (9H, s).

Compound 3:(S,S)-2-Allyl-2-chloro-3,4-dimethyl-5-phenyl-[1,3,2]oxazasilolidine wasprepared using a modified procedure of Leighton et al., J. Am. Chem.Soc. 125:9596 (2003) and WO 03/074534. The entire procedure wasperformed under an anhydrous closed system with minimized and briefmoments of exposure to the atmosphere. An oven-dried 5 L 4-neck flaskwith gas inlet, overhead stirring, and addition funnel was charged firstwith allyltrichlorosilane (419.5 g, 2.39 moles) and then with 2 Lanhydrous CH₂Cl₂. The solution was cooled under argon to 0° C. in anice/salt bath. Triethylamine (485 g, 4.79 moles) was added via additionfunnel, maintaining the temperature at 0° C. At this point the reactionwas a transparent amber color. S,S-pseudoephedrine (359 g, 2.17 moles)was added portionwise using a solid addition funnel over a period of 2h, maintaining an internal temperature <15° C. The addition funnel wasflushed with 200 mL CH₂Cl₂ into the reaction. Over time, paste-likesemi-granular solids formed (Et₃NCl). The light brown viscous slurry wasstirred for ca. 2 h on ice, then the ice bath was removed, therebyallowing the reaction to reach room temperature over a period of severalhours. Approximately 12 h later, most of the CH₂Cl₂ was removed bydistillation while maintaining the system under argon. 1 L anhydroushexane was added, and solvent was distilled again. 1 L anhydrous pentanewas then added and the mixture was stirred at room temperature for ca. 1h under argon. The previously paste-like sludge that originally formedin CH₂Cl₂ became more granular, first upon hexane addition, and thenespecially after pentane addition. The solution was light amber brown incolor. In portions, and by use of gentle argon pressure, pentane andsoluble product were transferred out of the reaction flask through aglass wool filter and PFA tube into second dry 1 L, 3-necked roundbottom flask equipped with a distillation head and magnetic stirring. Inalternation with solution transfer, pentane was distilled out of thissecond flask leaving behind the concentrated oily product. This processwas repeated, washing the Et₃NCl residue two times each with 500 mLpentane, and transferring the washes to the second flask under an argonatmosphere. The product was collected by distillation in fractions undera vacuum of ca. 10 torr. Ca. 56.3 g (9.7%) was collected in a forerunthat possibly contained up to ca. 3% allyltrichlorosilane. The mainfraction, purified(S,S)-2-allyl-2-chloro-3,4-dimethyl-5-phenyl-[1,3,2]oxazasilolidine, wasobtained in a quantity of 413.3 g (71%), bp=140-144 @ ca. 10 torr, as amildly viscous liquid light yellow at the time of collection, but whichbecame orange over several days under refrigeration and tightseptum/parafilm sealing. The material was stored under refrigeration,was not characterized, and was used without further purification in thesubsequent allylation reaction.

Compound 4: A 5 L, 4-neck flask equipped with thermometer and magneticstirring, was dried in an oven and maintained under argon while 2 Lanhydrous CH₂Cl₂ was added, followed byN′-(2,2-dimethyl-propylidene)-hydrazinecarboxylic acid benzyl ester (220g, 939 mmoles). The mixture was chilled and stirred in large, ca. 10 galice/brine bath at 2-3° C.(S,S)-2-allyl-2-chloro-3,4-dimethyl-5-phenyl-[1,3,2]oxazasilolidine (363g, 1.355 moles) was added to the flask over ca. 30 min using a cannulaand assistance with gentle argon pressure. The originally pale yellowsolution became a transparent light orange, while the temperatureremained at 2-3° C. The reaction was allowed to warm to room temperatureon its own accord and was monitored by quenching a small aliquot inCH₃OH and analyzing by TLC (2:1 hexanes:EtOAc). 36 hours afterinitiation, TLC analysis indicated ca. 90% completion. The mixture waschilled to 5° C., an additional 48 g (0.179 mmoles) oxazasilolidine wereadded, and the mixture was allowed to warm to room temperature. Ca. 8hours later, the reaction was again cooled to 5° C. and quenched with apre-chilled solution of 100 g K₂CO₃ in 100 mL deionized water over aperiod of ca. 1 h. During the quench, an exotherm raised the temperatureto as high as 17° C. Approximately 100 mL additional deionized water wasadded. The solution became a transparent blue-green in appearance. At notime during the reaction or quench was there any apparent evolution ofgas. After 4 days of stirring at room temperature, the solution waslight yellow in color, and after 5 days, the organic phase had gelledbut remained light yellow in color. The mixture was chilled to ca 15° C.(later apparent that this is not necessary), ca. 1.25 L hexanes wasadded, and the gel was partially collapsed and broken up with overheadstirring. The supernatant was siphoned off and filtered through glasswool while alternately adding ca. 1 L portions to the white gelremaining in the flask. Additional hexanes were also added to thesupernatant to precipitate pseudoephedrine as a white solid and tocollapse any remaining gel. The hexane extracts were combined andsolvent was removed in vacuo. Overall, through extractions of gel,hexanes-induced precipitation of pseudoephedrine, and filtrationsthrough glass wool and a glass-fritted Büchner funnel, approximately 5-6L hexanes was used to obtain the crude allyl hydrazide as a yellow oil.In batches, this crude material was treated with ca. three volumes ofhexanes, allowed to stand at room temp, decanted from precipitatedpseudoephedrine, and transferred to a separatory funnel. The productsolution was isolated from residual pseudoephedrine by washing thricewith ca. 25 mL each 1.4 N HCl (much color also removed), followed bywashes with 10% K₂CO₃ and brine. The hexanes solution of crude productwas dried over solid anhydrous Na₂SO₄, and solvent was removed in vacuoto give a light-yellow colored viscous oil. TLC (2:1 hexanes:EtOAc)showed desired product R_(f)=0.4; two impurities at 0.25 and 0.31 (5-10%each, one of them probably starting material), and a few less prominentimpurities above R_(f)=0.4. Pseudoephedrine previously detected at thebaseline was now absent. A total of 196.8 g (75.8%)(R)—N′-(1-tert-butyl-but-3-enyl)-hydrazinecarboxylic acid benzyl esterwas isolated as an amber oil after removing trace solvent on a vacuumpump. The original gel, now somewhat condensed and granular, wasextracted with ca. 2 L boiling hexanes. Solids were filtered, hexaneswere stripped, and the oil was decanted from precipitatedpseudoephedrine, yielding ca. 18 g. This material was combined withresidues from the primary product batch above (ca. 3-4 g), and washedwith 1.4 N HCl, aq. K₂CO₃, and brine as before. The hexanes werestripped to yield 20.6 grams additional product, as an amber oil whichby TLC appears identical to the first batch. A 10.1 g portion of(R)—N′-(1-tert-butyl-but-3-enyl)-hydrazinecarboxylic acid benzyl esterwas chromatographed quantitatively on silica gel to yield 8.88 g (88%yield for chromatography) pure product as a clear, off-white oil.R_(f)=0.43 (2:1 hexanes:ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 7.4(5H, br s), 6.2 (1H, br s), 6.0 (1H, br s), 5.15 (2H, s), 5.1 (2H, s),4.1 (1H, br), 2.7 (1H, m), 2.4 (1H, br d), 2.0 (1H, m), 0.95 (9H, s);ee=98.1%, AD-H column; [α]²⁵ ₅₈₉ −42.5° (c 2.18, CH₃OH).

Compound 5: (R)—N′-(1-tert-butyl-but-3-enyl)-hydrazinecarboxylic acidbenzyl ester (110 g, 398 mmoles) and 500 mL of methylene chloride wereadded to a 2 L round bottom flask with a thermometer and magneticstirrer. A solution of K₂CO₃ (82.51 g, 597 mmol in 200 mL deionizedwater) was subsequently added, and the flask was cooled to ca. 10° C.Neat 3,5-dimethyl benzoyl chloride (73.82 g, 437.8 mmol) was then addedslowly over a period of 20 min. 1 L methylene chloride was used to rinseresidual acid chloride into the flask and to dilute the reactionmixture. The temperature was not allowed to exceed 15° C. during theaddition. The reaction was stirred overnight, first in an ice bath andthen at room temperature. TLC analysis at ca. 16 hours indicated thatthe reaction was complete with residual acid chloride; the mixture wasstirred for a total of ca. 40 hours. The reaction mixture was pouredinto a 2 L separatory funnel. The organic layer was collected andcombined with a small CH₂Cl₂ backwash of the aqueous layer. The organicphase was washed once more with 10% K₂CO₃, washed with brine, and thendried over solid MgSO₄/Na₂SO₄. The mixture was filtered from salts, andsolvent was removed in vacuo to obtain a granular light beige solid. Thecrude product was suspended and stirred in 300 mL of 2:1 hexanes:etherand filtered on a Büchner funnel to substantially remove residual3,5-dimethylbenzoyl chloride. The collected solids were washed fourtimes more with a combined total of ca. 1200 mL 2:1 hexanes:ether,thereby providing a white granular solid. The product was collected andallowed to dry in the air to yield 144.55 g(R)—N′-(1-tert-butyl-but-3-enyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylicacid benzyl ester, yield=88.9%. A 30 g sample of was dissolved in 205 mLboiling methanol and allowed to crystallize at room temperature. Theresultant flocculant material was washed in a Büchner with 50 mL CH₃OHto give 22.2 g, mp=146° C., (ee>99.9%). An analytical sample of(R)—N′-(1-tert-butyl-but-3-enyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylicacid benzyl ester was obtained by twice-recrystallizing this materialfrom boiling CH₃OH followed by Kugelrohr short-path distillation andcollection as a solid to give a pure white material: mp=145.5-147° C.R_(f)=0.12 (10:1 hexanes:ethyl acetate); ¹H NMR (400 MHz, CDCl₃):

7.5-7.2 (m, 9H, φ+NH), 7.2-6.8 (m), 6.4 (S), 6.3 (S), 5.95 (br, 5H,benzylic+C═C), 5.4-4.6 (m, 9H, allylic N—CH, φ-CH3), 3.65 (br), 2.5 (m),2.35 (S), 2.3 (S), 1.2-0.8 (m, 9H, —C(CH₃)₃); [α]²⁵ ₅₈₉ −12.8° (c 2.01,CHCl₃).

Compound 6(R)—N′-(1-tert-butyl-but-3-enyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic acid benzyl ester (13.5 g, 33 mmoles) was suspended in 100 mLglacial acetic acid at room temperature. 10% Palladium on charcoal (0.24g) were added as a slurry in 4.4 g acetic acid. The gray suspension wasshaken for 90 minutes at 10-30 psi on a Parr hydrogenator, monitoringthe reaction by TLC. The catalyst was allowed to settle, and thereaction solution was removed with a pipette and passed through flutedfilter paper (Schleicher & Schuell 597½, 125 mm dia). The catalystresidue was washed with acetic acid and washes were passed throughfilter paper to give a combined total mass of 156 g acetic acid andproduct. The mixture was chilled on ice and ca. 600 mL deionized waterwas added. Over a period of 30 minutes, product oiled out and thencrystallized. This was filter through paper, dried in air, and collecton paper to yield 7.9 g of a light yellow solid. Over several hours,0.52 g additional product precipitated out of the filtrate to give atotal yield of 8.42 g (92.2%) of (R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-hydrazide; R_(f)=0.5 (1:1 hexanes:ethyl acetate);[α]²⁵ ₅₈₉ +7.63 (c 1.98, CH₃OH), ¹H NMR (400 MHz, CDCl₃) δ 7.05 (1H, s),7.02 (2H, s), 4.6+3.5 (1H, 2 d), 4.1 (2H, s, NH₂), 2.38+2.37 (6H, 2 s),1.1-2.1 (4H, m), 1.0+0.9 (9H, 2 s), 1.0+0.98 (3H, 2 t), 2 distinctconformers.

Compound 7 (title compound): (R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-hydrazide (35 g, 126.6 mmoles) was dissolved in100 mL methylene chloride in a 500 mL round bottom flask with magneticstirring. Aqueous K₂CO₃ (26.25 g, 189.9 mmoles, solution in 75 mLdeionized water) was added and the mixture was stirred on ice. Solid2-ethyl-3-methoxybenzoyl chloride (27.67 g, 139.3 mmoles) was added andrinsed into the flask using 25 mL CH₂Cl₂. The mixture was stirred on icefor ca. 40 h, allowing the bath to warm to room temperature. Additionalmethylene chloride and water were added as needed to aid manipulation.The organic layer was separated and dried over MgSO₄. Ca. 5 g activatedcharcoal was added and the salt and carbon were removed by filtrationthrough paper. The solvent was evaporated in vacuo, first on a rotaryevaporator and then under high vacuum, providing 57.8 g crude product,containing traces of CH₂Cl₂. The crude material was triturated firstwith 3×100 mL portions of ether (2% ethanol) and then with 1×100 mLportion of 1:1 hexanes:ether in a fritted glass Büchner funnel (poresize ASTM 40-60 C, Pyrex). The product was then washed with 2×200 mLdeionized water with thorough mixing and allowed to dry in air.(R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide (alsoreferred to as (R)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide) wascollected as a bright white powdery solid (36.51 g, 65.7% yield, quitepure and very high ee by HPLC and chiral HPLC, one spot by TLC, 1:1hexanes:ether). Evaporation of the combined organic washes yielded ca.20 g material, which was triturated first with 200 mL 2:1 hexanes:etherand then with 2×100 mL 2:1 hexanes:ether in a fritted glass Büchner,pore size ASTM 40-60 C. The material was washed with 3×100 mL deionizedwater with thorough mixing and allowed to air dry. A second crop wascollected as a white powder (12.98 g, 23.4% yield, quite pure and highee by HPLC and chiral HPLC). A third crop was obtained in a similarmanner as an off-white solid (1.52 g, 2.7%, 98% pure, >99% ee). A finalsample was prepared by dissolving in warm methanol and filtering througha 0.45 micron syringe filter into a large crystallization dish. Thematerial was collected, pulverized with a spatula, and heated at 50-58°C. in a vacuum oven virtually until constant weight. HPLC analysisindicated 99.3% chemical purity and >99.9% ee R_(f)=0.28 (1:1hexanes:ethyl acetate); mp=162.2-162.8° C.; [α]²⁵ ₅₈₉ +12.9 (c 2.04,CH₃OH); ¹H NMR (400 MHz, CD₃OD) δ 7.0-7.1 (4H, m), 6.9 (1H, d), 6.1-6.4(1H, 2 d), 4.5-4.7 (1H, 2 d), 3.78 (3H, s), 2.3 (6H, s), 2.3 (1H, m),1.9 (2H, m), 1.55 (3H, m), 1.1-1.15 (9H, 2 s), 0.9-1.0 (6H, m).

Examples 2 to 29 were prepared using methodology described in Example 1.The percent enantiomeric excess (ee) was determined by chiral HPLC.Table 2 provides analytical data for examples 2 to 29.

Example Structure ee; % Name 2

>99 (R)-3,5-Dimethyl- benzoic acid N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide 3

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(2-methyl-benzoyl)- hydrazide 4

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(2-methoxy-benzoyl)- hydrazide 5

>98 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(2-fluoro-benzoyl)- hydrazide 6

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(2-chloro-benzoyl)- hydrazide 7

>99 (R)-3,5-Dimethyl- benzoic acid N′-(2- bromo-benzoyl)-N-(1-tert-butyl-butyl)- hydrazide 8

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(3-methyl-benzoyl)- hydrazide 9

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(3-methoxy-benzoyl)- hydrazide 10

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(3-chloro-benzoyl)- hydrazide 11

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(4-methyl-benzoyl)- hydrazide 12

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(4-ethyl-benzoyl)-hydrazide 13

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(4-methoxy-benzoyl)- hydrazide 14

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(4-chloro-benzoyl)- hydrazide 15

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(2,6-difluoro-benzoyl)- hydrazide 16

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(2,6-dichloro-benzoyl)- hydrazide 17

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert-butyl-butyl)-N′-(3,4-dimethoxy-benzoyl)- hydrazide 18

>98 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(3,5-difluoro-benzoyl)- hydrazide 19

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert-butyl-butyl)-N′-(3,5-dimethoxy-4-methyl- benzoyl)-hydrazide 20

 97 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(4-methyl-benzo[1,3] dioxole-5-carbonyl)- hydrazide 21

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(5-methyl-2,3-dihydro- benzo[1,4]dioxine-6- carbonyl)-hydrazide 22

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(5-ethyl-2,3-dihydro-benzo [1,4]dioxine-6- carbonyl)-hydrazide 23

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(naphthalene-1- carbonyl)-hydrazide 24

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(naphthalene-2- carbonyl)-hydrazide 25

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(thiophene-2-carbonyl)- hydrazide 26

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(2,5-dimethyl-furan-3- carbonyl)-hydrazide 27

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(2-chloro-pyridine-3- carbonyl)-hydrazide 28

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(6-chloro-pyridine-3- carbonyl)-hydrazide 29

>99 (R)-3,5-Dimethyl- benzoic acid N-(1-tert- butyl-butyl)-N′-(3-methoxy-2-methyl- benzoyl)-hydrazide

TABLE 2 Analytical Data Example ¹H NMR (solvent) MS MP; ° C. 2 (CDCl₃) δ7.88-7.0 (m, 8H), [M + H]⁺ 381.2533, 66-68 4.75 + 3.69 (2d, 1H), 2.4 +2.28 (2s, 6H), [M + Na]⁺ 403.2351 2.0-1.3 (mm, 4H), 1.15 + 1.02 (2s,9H), 0.94 (m, 3H) ppm 3 (CDCl₃) δ 7.62-6.77 (m, 7H), [M + H]⁺ 395.2685,137-138 4.75 + 3.72 (2d, 1H), 2.34 + 2.33 (2s, 6H), 2.02 (s, [M + Na]⁺417.2507 3H), 1.85-1.42 (mm, 4H), 1.19 + 1.14 (2s, 9H), 1.01 (m, 3H) ppm4 (DMSO-d₆) δ 10.07 + 9.98 (2s, NH), [M + H]⁺ 411.2637, 7.73-6.63 (m,7H), 4.54 + 4.39 (2d, 1H), [M + Na]⁺ 433.2459 3.91 + 3.77 (2s, 3H),2.36 + 2.28 (2s, 6H), 1.77-1.25 (mm, 4H), 1.1 + 0.98 (2s, 9H), 0.87 (m,3H) ppm 5 (DMSO-d₆) δ 10.48 + 10.35 (2s, NH), [M + H]⁺ 399.2436, 114-1167.61-6.68 (m, 7H), 4.52 + 4.37 (2d, 1H), [M + Na]⁺ 421.2253 2.25 (s,6H), 1.76-1.19 (mm, 4H), 1.02 + 0.83 (m, 12H) ppm 6 (CDCl₃) δ 7.84-6.6(m, 7H), [M + H]⁺ 415.2146, 138-140 4.74 + 3.72 (2d, 1H), 2.4 + 2.34(2s, 6H), [M + Na]⁺ 437.1962 1.97-1.44 (mm, 4H), 1.18 + 1.13 (2s, 9H),0.99 (m, 3H) ppm 7 (DMSO-d₆) δ 10.47 + 10.37 (2s, NH), [M + H]⁺459.1637, 145-147 7.62-6.13 (m, 7H), 4.52 + 4.35 (2d, 1H), [M + Na]⁺481.1453 2.28 + 2.26 (2s, 6H), 1.79-1.38 (mm, 4H), 1.07 + 0.85 (m, 12H)ppm 8 (CDCl₃) δ 7.72-7.09 (m, 7H), [M + H]⁺ 395.2685, 158-159 4.74 +3.68 (2d, 1H), 2.4 + 2.38 (2s, 6H), 2.29 (s, [M + Na]⁺ 417.2503 3H),2.0-1.45 (mm, 4H), 1.31 + 1.15 (2s, 9H), 1.02 (m, 3H) ppm 9 (DMSO-d₆) δ10.3 + 10.12 (2s, NH), [M + H]⁺ 411.2637, 110-111 7.48-6.8 (m, 7H),4.57 + 4.41 (2d, 1H), [M + Na]⁺ 433.2456 3.86 + 3.77 (2s, 3H), 2.35 +2.25 (2s, 6H), 1.78-1.29 (mm, 4H), 1.08 + 1.04 (2s, 9H), 0.98-0.88 (m,3H) ppm 10 (CDCl₃) δ 7.83-7.07 (m, 7H), [M + H]⁺ 415.2148, 163-1644.74 + 3.69 (2d, 1H), 2.4 + 2.31 (2s, 6H), [M + Na]⁺ 437.1963 2.0-1.45(mm, 4H), 1.15 + 1.09 (2s, 9H), 1.02-0.94 (m, 3H) ppm 11 (CDCl₃) δ8.08-7.08 (m, 7H), [M + H]⁺ 395.2687, 60-62 4.74 + 3.68 (2d, 1H), 2.46 +2.40 (2s, 6H), [M + Na]⁺ 417.2511 2.34 + 2.28 (2s, 3H), 1.74-1.31 (mm,4H), 1.15 + 1.02 (2s, 9H), 0.95 (m, 3H) ppm 12 (DMSO-d₆) δ 10.21 + 10.04(NH, 2s) d [M + H]⁺ 409.2844, 111-112 7.78-6.93 (m, 7H), 4.54 + 4.39(2d, 1H), [M + Na]⁺ 431.2670 2.60 (m, 2H), 2.32 + 2.21 (2s, 6H),1.84-1.21 (mm, 4H), 1.15 (t, 3H), 1.05-0.84 (m, 12H) ppm 13 (DMSO-d₆) δ10.16 + 9.98 (2s, NH), [M + H]⁺ 411.2640, 65-67 7.88-6.97 (m, 7H),4.57 + 4.41 (2d, 1H), [M + Na]⁺ 433.2458 3.87 + 3.8 (2s, 3H), 2.35 +2.24 (2s, 6H), 2.02-1.21 (mm, 4H), 1.08 + 1.02 (2s, 9H), 0.93-0.86 (m,3H) ppm 14 (CDCl₃) δ 8.38-7.06 (m, 7H), [M + H]⁺ 415.2136, 87-89 4.74 +3.7 (2d, 1H), 2.41 + 2.28 (2s, 6H), [M + Na]⁺ 437.1961 1.65-1.31 (mm,4H), 1.14 + 1.02 (2s, 9H), 0.93 (m, 3H) ppm 15 (DMSO-d₆) δ 10.62 + 10.54(NH, 2s) d [M + H]⁺ 417.2335, 180-182 7.45-6.96 (m, 6H), 4.50 + 4.35(2d, 1H), [M + Na]⁺ 439.2160 2.29 + 2.22 (2s, 6H), 1.70-1.15 (mm, 4H),1.0-0.80 (m, 12H) ppm 16 (DMSO-d₆) δ 10.53 (s, 1H), [M + H]⁺ 449.1730,185-187 7.53-7.35 (m, 3H), 7.08 (s, 2H), 7.03 (s, 1H), [M + Na]⁺471.1546 4.64 (d, 1H), 2.31 + 2.28 (2s, 6H), 1.89-1.42 (mm, 4H), 1.06(s, 9H), 0.93-0.86 (m, 3H) ppm 17 (DMSO-d₆) δ 10.08 + 9.89 (NH, 2s) d[M + H]⁺ 441.2732, 121-123 7.75-6.87 (m, 6H), 4.53 + 4.38 (2d, 1H), [M +Na]⁺ 463.2545 3.88 + 3.72 (m, 6H), 2.32-2.21 (m, 6H), 1.81-1.21 (mm,4H), 1.05-0.82 (m, 12H) ppm 18 (DMSO-d₆) δ 10.40 + 10.21 (NH, 2s) d [M +H]⁺ 417.2337, 192-194 7.57-6.97 (m, 6H), 4.53 + 4.38 (2d, 1H), [M + Na]⁺439.2155 2.31 + 2.22 (2s, 6H), 1.71-1.24 (mm, 4H), 1.03 + 0.99 (2s, 9H),0.88-0.76 (m, 3H) ppm 19 (DMSO-d₆) δ 10.16 + 9.98 (2s, 1H), [M + H]⁺455.2913, 7.37-6.51 (m, 5H), 4.58 + 4.41 (2d, 1H), [M + Na]⁺ 477.27283.91 + 3.86 (2s, 3H), 3.78 (s, 3H), 2.36 + 2.26 + 2.13 + 2.10 (4s, 6H),2.00 (s, 3H), 1.80 + 1.30 (m, 4H), 1.09 + 1.06 (2s, 9H), 0.99 + 0.89(2t, 3H) ppm 20 (CDCl3) δ 7.26-6.43 (m, 5H), [M + H]⁺ 439.2609, 6.06 +5.99 (2s, 2H), 4.72 + 4.60 (2d, 1H), [M + Na]⁺ 461.2407 2.45-2.32 (m,6H), 1.88 (s, 3H), 1.99-1.44 (m, 4H), 1.17-0.97 (m, 12H) ppm 21(DMSO-d₆) δ 10.25 + 10.10 (s, 1H), 70 7.13 (s, 2H), 7.05 (s, 1H), 6.70(d, 1H), 6.45 + 6.36 (2d, 1H), 4.56 + 4.36 (2d, 1H), 4.22-3.53 (mm, 4H),2.28 (s, 6H), 1.80-1.37 (mm, 4H), 1.65 + 1.58 (2s, 3H), 1.06 (s, 9H),0.96 + 0.88 (2t, 3H) ppm 22 (CDCl₃) δ 7.08-6.22 (m, 5H), [M + H]⁺467.2905, 128-129 4.74 + 3.69 (2d, 1H), 4.3-4.13 (m, 4H), [M + Na]⁺489.2721 2.39 + 2.34 (2s, 6H), 2.14-1.95 (m, 2H), 1.44-1.29 (mm, 4H),1.18 + 1.12 (2s, 9H), 1.0 (m, 6H) ppm 23 (CDCl₃) δ 9.2-7.08 (m, 10H),[M + H]⁺ 431.2691, 173-174 4.81 + 3.76 (2d, 1H), 2.42 + 2.32 (2s, 6H),[M + Na]⁺ 453.2512 2.08-1.45 (mm, 4H), 1.25-1.02 (m, 12H) ppm 24 (CDCl₃)δ 8.43-6.94 (m, 10H), [M + H]⁺ 431.2752, 91(glass)-134 4.78 + 3.73 (2d,1H), 2.41 + 2.29 (2s, 6H), [M + Na]⁺ 453.2512 2.03-1.31 (mm, 4H),1.18-0.94 (m, 12H) ppm 25 (DMSO-d₆) δ 10.25 + 10.08 (2s, 1H), [M + H]⁺387.2104, 7.95-6.91 (m, 6H), 4.58 + 4.39 (2dd, 1H), [M + Na]⁺ 409.19232.31 + 2.24 (2s, 6H), 1.90-1.25 (m, 4H), 1.08 + 1.01 (2s, 9H), 0.96-0.84(m, 3H) ppm 26 (DMSO-d₆) δ 9.78 + 9.56, 7.14 + 7.08 (2s, [M + H]⁺399.2644 1H), 7.03 + 6.98 (2s, 1H), 6.29 + 2.24 (2s, 1H), 4.53 + 4.37(2d, 1H), 2.25 (s, 6H), 2.21 + 2.17 (2s, 6H), 1.78-1.23 (m, 4H), 1.05 +1.00 (2s, 9H), 0.98 + 0.86 (2t, 3H) ppm 27 (DMSO-d₆) δ 10.59 + 10.51(2s, 1H), [M + H]⁺ 416.2103, 8.48 (dd, 1H), 7.45 (dd, 1H), 7.13 (s, [M +Na]⁺ 438.1922 2H), 7.04 (s, 1H), 6.73 (dd, 1H), 6.62 (dd, 1H), 4.56 +4.41 (2d, 1H), 2.30 (s, 6H), 1.79-1.27 (m, 4H), 1.09 + 1.05 (2s, 9H),0.97 + 0.91 (2t, 3H) ppm 28 (DMSO-d₆) δ 10.54 + 10.38 (2s, 1H), [M + H]⁺416.2102, 8.39-7.00 (m, 6H), 4.58 + 4.42 (2d, 1H), [M + Na]⁺ 438.19242.25 (s, 6H), 1.86-1.30 (m, 4H), 1.08 + 1.04 (2s, 9H), 1.00 + 0.89 (2t,3H) ppm 29 (DMSO-d₆) δ 10.39 + 10.22 (2s, NH), [M + H]⁺ 425.2804,141-143 7.16-7.0 (m, 5H), 6.45 + 6.33 (2d, 1H), [M + Na]⁺ 447.26164.57 + 4.39 (2d, 1H), 3.85 + 3.78 (2s, 3H), 2.36 + 2.29 (2s, 6H),1.80-1.46 (mm, 4H), 1.09 + 1.06 (2s, 9H), 0.96-0.89 (m, 3H) ppm

Example 30

Synthesis

Compound 1: (R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-hydrazide was prepared as described in Example 1.

Compound 2: Compound 2 was prepared from the corresponding carboxylicacid. Thionyl chloride (1.24 g, 10.42 mmoles) was added to a solution of(S)-(+)-2-(6-methoxy-naphthalen-2-yl)-propionic acid (Naproxen, 2 g,8.69 mmoles) in 8.2 mL chloroform in a round bottom flask equipped withmagnetic stirring. A drop of DMF was added and the mixture was refluxedfor 3 hours. Chloroform and excess thionyl chloride were distilled fromthe reaction mixture while methylene chloride was added. Evaporation ofresidual solvent yielded (S)-2-(6-methoxy-naphthalen-2-yl)-propionylchloride (1.918 g, 88.8% yield). R_(f)=0.2 (1:1 hexanes:ethyl ether). ¹HNMR (400 MHz, CDCl₃) δ 9.98 (br, 1H), 7.78 (t, 2H), 7.36 (d, 1H), 7.18(d, 1H), 7.14 (m, 2H), 4.25 (q, 1H), 3.93 (s, 3H), 1.68 (d, 3H).

Compound 3 (title compound): (R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-hydrazide (e.e estimated at >99% based onprecursor, 1.1 g, 3.98 mmoles) was dissolved in 10 mL methylenechloride. Aqueous K₂CO₃ solution (0.907 g, 6.57 mmoles in 2.5 mL) wasadded to the reaction mixture.(S)-2-(6-methoxy-naphthalen-2-yl)-propionyl chloride (1.09 g, 4.38mmoles) was added and the reaction was stirred for 24 hours andmonitored by TLC. Additional methylene chloride and water were added asneeded to aid manipulation. The organic layer was separated, dried overNa₂SO₄, filtered, and solvent was removed in vacuo to provide crudeproduct: mp=98° C. (glass)-123° C. The crude material was trituratedthrice with 2:1 hexanes:ether to provide 1.554 g, 80.1% yield of(R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(S)-[2-(6-methoxy-naphthalen-2-yl)-propionyl]-hydrazide,mp=98° C. (glass)-122° C. The triturated material was crystallized fromisopropanol at 35° C. to give crystalline product. ¹H NMR spectra of allthree lots were identical. R_(f)=0.15 (1:1 hexanes:ethyl ether);mp=158-160° C. (heating commenced at 150° C.); mp=156-164° C. (heatingcommenced at 25° C.); [α]²⁵ ₅₈₉ +95.8° (c 2.02, CH₃OH); ¹H NMR (400 MHz,DMSO-d₆) δ 9.96+9.82 (NH, 2 s), 7.71+7.58 (2H, 2 d), 7.63 (1H, s),7.49+7.31 (1H, 2 d), 7.26 (1H, s), 7.13+6.77 (1H, 2 d), 7.06 (2H, s),6.68+6.30 (1H, 2 s), 4.31 (m, 1H), 3.88+3.85 (3H, 2 s), 3.58 (1H, m),2.30+1.83 (6H, 2 s), 1.64-1.03 (4H, m), 1.00 (3H, m), 0.87+0.84 (9H, 2s), 0.63 (3H, t); ¹³C NMR (100 MHz, CDCl₃) δ 173.9, 172.2, 157.8, 137.3,133.7, 130.8, 129.1, 128.8, 127.4, 125.9, 125.8, 123.7, 123.3, 119.2,119.1, 105.5, 62.7, 55.3, 45.1, 35.0, 28.5, 27.6, 26.9, 21.2, 17.5,14.2; HRMS (ESI) m/z calcd for C₃₁H₃₉N₂O₃ [M−H]⁻ 487.2966, found487.2949, calcd for C₃₁H₄₀ClN₂O₃ [M+Cl]⁻ 523.2733, found 523.2718, Anal.Calcd for C₃₁H₄₀N₂O₃: C, 76.19; H, 8.25; N, 5.73; 0, 9.8. Found: C,76.01; H, 8.35; N, 5.70.

In order to determine the stereochemical course of the asymmetricallylation reaction in Example 1, the x-ray crystal structure of(R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(S)-[2-(6-methoxy-naphthalen-2-yl)-propionyl]-hydrazidewas determined. This experiment established the absolute configurationof the carbon bearing the tert-butyl group and n-propyl group is R.

Example 31

Synthesis

Compound 1: (E)-N′-(2,2-dimethyl-propylidene)-hydrazinecarboxylic acidbenzyl ester was prepared as described in Example 1.

Compound 2:(R,R)-2-allyl-2-chloro-3,4-dimethyl-5-phenyl-[1,3,2]oxazasilolidine wasprepared using methodology described in Example 1 usingR,R-pseudephedrine.

Compound 3: (S)—N′-(1-tert-butyl-but-3-enyl)-hydrazinecarboxylic acidbenzyl ester was prepared using methodology described in Example 1.Analytical data: R_(f)=0.46 (2:1 hexanes:ethyl acetate); mp=69-71° C.;¹H NMR (400 MHz, CDCl₃) δ 7.4 (br s, 5H), 6.2 (br s, 1H), 6.0 (br s,1H), 5.15 (s, 2H), 5.1 (s, 2H), 4.1 (br, 1H), 2.7 (m, 1H), 2.4 (br d,1H), 2.0 (m, 1H), 0.95 (s, 9H); ee=98.2%, ADH column; [α]²⁵ ₅₈₉ +39.30(c=2.03, CHCl₃).

Compound 4:(S)—N′-(1-tert-butyl-but-3-enyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic acid benzyl ester was prepared using methodology described inExample 1. Analytical data: R_(f)=0.43 (2:1 hexane:ethyl acetate);mp=150-151.5° C.; ¹H NMR (400 MHz, CDCl₃) δ 7.45-6.88 (m, 7H), 6.44+6.28(2 s, 1H), 5.96 (br, 1H), 5.38-4.68 (m, 5H), 3.69 (br, 1H), 2.68-2.43(m, 2H), 2.36+2.29 (2 s, 6H), 1.13+1.02+0.94 (3 s, 9H); [α]²⁵ ₅₈₉ +12.5°(c=2.01, CHCl₃).

Compound 5: (S)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-hydrazide was prepared using methodologydescribed in Example 1. Analytical data: R_(f)=0.5 (1:1 hexanes:ethylacetate); mp=112-112.8° C.; ¹H NMR (400 MHz, CDCl₃) δ 7.05 (s, 1H), 7.02(s, 2H), 4.6+3.5 (2d, 1H), 2.38+2.37 (2 s, 6H), 1.2-2.1 (m, 4H), 1.1+1.0(2 s, 9H), 1.05+0.98 (2 t, 3H) 2 distinct conformers; [α]²⁵ ₅₈₉ −7.97 (c2.14, CH₃OH).

Compound 6 (title compound): (S)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide wasprepared using methodology described in Example 1. Analytical data:R_(f)=0.28 (1:1 hexanes:ethyl acetate); mp=156.5-157.5° C.; [α]²⁵ ₅₈₉−13.39° (c 2.05, CH₃OH); ¹H NMR (400 MHz, DMSO-d₆) δ 10.4+10.26 (s, 1H),7.18-7.14 (m, 3H), 7.08 (d, J=7.2 Hz, 1H), 7.03 (t, J=8.4 Hz, 1H),6.34+6.21 (d, J=6.8 Hz, 1H), 4.57+4.38 (d, J=8.4 Hz, 1H), 3.78 (s, 3H),2.29 (s, 6H), 2.27-2.23 (m, 1H), 1.89-1.79 (m, 2H), 1.59-1.38 (m, 3H),1.09+1.06 (s, 9H), 0.95+0.87 (t, J=6.8 Hz, 6H); ¹³C NMR (100 MHz,DMSO-d₆) δ 173.40, 168.24, 157.64, 137.18, 136.98, 136.50, 130.89,130.73, 130.20, 126.96, 125.28, 124.83, 119.37, 118.95, 112.41, 62.29,56.11, 35.45, 28.71, 27.56, 21.24, 20.15, 15.15, 14.68; HRMS (ESI) m/z,calcd for C₂₇H₃₉N₂O₃ [M+H]⁺ 439.2955, found 439.295, calcd forC₂₇H₃₈NaN₂O₃ [M+Na]⁺ 461.2774, found 461.2765; Anal. Calcd forC₂₇H₃₈N₂O₃: C, 73.94; H, 8.73; N, 6.39; O, 10.94. Found: C, 73.87; H,8.94; N, 6.38; ee: >99.9%; Regis (S,S) ULMO, 98:2 mixture ofhexanes:methanol at a flow rate of 1 mL/min.

Examples 32 to 47 were prepared using methodology described in Example31. The percent enantiomeric excess (ee) was determined by chiral HPLC.

Example Structure % ee Name 32

>99 (S)-(3,5-Dimethyl-benzoic acid N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide 33

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide 34

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide 34

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-chloro-benzoyl)-hydrazide 36

>99 (S)-3,5-Dimethyl-benzoic acid N′-(2-bromo-benzoyl)-N-(1-tert-butyl-butyl)-hydrazide

37

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide 38

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide 39

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-ethyl-benzoyl)-hydrazide 40

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2,6-difluoro-benzoyl)-hydrazide 41

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide 42

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3,5-dimethoxy-4-methyl- benzoyl)-hydrazide 43

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(thiophene-2-carbonyl)- hydrazide 44

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2,5-dimethyl-furan-3-carbonyl)- hydrazide 45

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-chloro-pyridine-3-carbonyl)- hydrazide 46

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(6-chloro-pyridine-3-carbonyl)- hydrazide 47

>99 (S)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)- hydrazide

Example 48

Synthesis

Compound 1: t-Butyl carbazate is commercially available.

Compound 2: Pivaldehyde (52.1 g, 454 mmoles, ca. 75% solution int-butanol) was dissolved in 120 mL methanol in a 1 L, 3-necked roundbottom flask with reflux condenser, thermometer, and addition funnel.Glacial acetic acid (1 mL) was added followed by controlled addition oft-butyl carbazate (50 g, 378 mmoles), without removal of heat of thereaction. The mixture was stirred for 4 hours, while monitoring by TLC.Upon completion of the reaction, the solvent was removed in vacuo, theresidue was taken up in pentane, and the pentane was evaporated. Theresultant oil crystallized upon standing to provideN′-(2,2-dimethyl-propylidene)-hydrazinecarboxylic acid tert-butyl ester(75.7 g, 93%). R_(f)=0.42 (2:1 hexanes:ethyl acetate, 1% acetic acid);¹H NMR (400 MHz, CDCl₃) δ 7.6 (br s, 1H), 7.1 (s, 1H), 1.5 (s, 9H), 1.1(s, 9H) ppm.

Compound 3:(R,R)-2-Allyl-2-chloro-3,4-dimethyl-5-phenyl-[1,3,2]oxazasilolidine wasprepared using methodology described in Example 1 usingR,R-pseudephedrine.

Compound 4: N′-(2,2-Dimethyl-propylidene)-hydrazinecarboxylic acidtert-butyl ester (12 g, 59.9 mmoles) was charged to a dried, 3-neck, 1 Lround bottom flask with thermometer, magnetic stirrer, and nitrogenatmosphere. Anhydrous methylene chloride (200 mL) was charged to theflask using a catheter. The mixture was chilled to 0° C. with an icebath.(R,R)-2-Allyl-2-chloro-3,4-dimethyl-5-phenyl-[1,3,2]oxazasilolidine(24.07 g, 89.87 mmoles) was subsequently added to the flask under inertconditions. Within minutes, the originally light yellow solution turnedtransparent dark yellow. The reaction was stirred for 6 hr at 0° C.,then allowed to warm to room temperature, and stirred overnight, whilebeing monitored by TLC. The reaction was quenched by adding ca. 25 mLmethanol, resulting in a lightening of color. The solution wasconcentrated and the residue was diluted with 100 mL of ethyl acetateand 100 mL of water. The phases were separated and the aqueous layer wasextracted with ethyl acetate twice. The organic phases were combined,washed with brine, dried over Na₂SO₄ and concentrated. The resulting oilwas purified by column chromatography using 10% ethyl acetate inhexanes. Purified (S)—N′-(1-tert-butyl-but-3-enyl)-hydrazinecarboxylicacid tert-butyl ester was obtained as an oil (2.87 g, 24.4% yield). Animpure fraction was also collected (3.17 g). R_(f)=0.44 (5:1hexanes:ethyl acetate, 12 visualization); ¹H NMR (400 MHz, CDCl₃) δ 6.07(br, 1H, NH), 5.85 (m, 1H), 5.0 (dd, 2H), 2.6 (m, 1H), 2.33 (m, 1H), 2.3(m, 1H), 1.4 (s, 9H), 0.9 (s, 9H).

Compound 5: (S)—N′-(1-tert-Butyl-but-3-enyl)-hydrazinecarboxylic acidtert-butyl ester (2.34 g, 9.65 mmoles) was dissolved in methanol (50 mL)in a Parr bottle. 10% Pd/C (70 mg) was added as a slurry in water (1.6mL), and the mixture was shaken on a Parr hydrogenator for 4.5 h with astarting pressure of 55 psi. The pressure decrease was monitored as ameasure of the reaction progress, indicating that the reaction waslikely complete after 30 minutes. The catalyst was allowed tosubstantially settle, and the supernatant was removed with a pipette,passed through a 0.45 micron syringe filter, and analyzed by TLC.Solvent was removed in vacuo to yield 2.19 g (92.7%) of(S)—N′-(1-tert-butyl-butyl)-hydrazinecarboxylic acid tert-butyl ester asa faintly pale yellow oil. R_(f)=0.48 (5:1 hexanes:ethyl acetate, 12visualization), ¹H NMR (400 MHz, CDCl₃) □ 6.0 (br, 1H), 4.0 (br, 1H),2.5 (d, 1H), 1.5 (s, 9H), 1.1 (m, 2H), 1.6 (br, 2H), 0.94 (s, 9H), 0.94(t, 3H).

Compound 6 (title compound):(S)—N′-(1-tert-Butyl-butyl)-hydrazinecarboxylic acid tert-butyl ester (1g, 4.09 mmoles) was dissolved in ca. 3 mL methylene chloride in a vialwith a magnetic stir bar. A solution of 0.8 g K₂CO₃ (5.79 mmoles) in ca.2 mL water was added, followed by 0.81 g (4.80 mmoles)3,5-dimethylbenzoyl chloride. The reaction was stirred overnight, firston ice, and then allowed to warm to room temperature. Several mL eachwater and methylene chloride were then added to dissolve all solids. Theaqueous layer was removed and the organic layer was dried over solidMgSO₄. TLC indicated that the reaction was ca. 95% complete. The mixturewas filtered through some glass wool into a vial. Solvent was evaporatedwith a stream of N₂, chasing with 3:1 hexanes:ether and pentane. Theresidue was manipulated with a spatula to initiate crystallization, andafter complete solidification, was triturated thrice with 2-3 mL pentanein a small fritted glass funnel. After drying in air, 300 mg (19.5%)(S)—N′-(1-tert-Butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylicacid tert-butyl ester was obtained, indicated by TLC to be quite pure,chiral HPLC indicated e.e=93.3%. The product was dried in a vacuum ovenat 50° C. for melting point determination. R_(f)=0.42 (4:1 hexanes:ethylacetate, UV (strong), 12 (weak) visualization); mp=115.5-117.5° C.;[α]²⁵ ₅₈₉ −30.18 (c 2.02, CH₃OH), ¹H NMR (400 MHz, CDCl₃) δ 7.15-7.03(m, 3H), 6.07+6.00 (s+d, 1H), 4.52+4.40 (2 dd, 1H), 2.32+2.28+2.23 (3 s,6H), 1.85-0.89 (m, 25H).

Example 49

Compound 1: (R)—N′3,5-Dimethyl-benzoic acidN-(1-tert-butyl-but-3-enyl)-hydrazide was prepared as described inExample 1.

Compound 2 (title compound): To a solution of (R)-3,5-dimethyl-benzoicacid N-(1-tert-butyl-butyl)-hydrazide (400 mg, 1.44 mmol) in THF (10 mL)was added t-boc anhydride (629 mg, 2.88 mmol). The reaction was refluxedfor 40 h, cooled, diluted with ether (20 mL), washed with H₂O, and driedover anhydrous Na₂SO₄. The solvent was evaporated in vacuo and theresidue was purified by flash chromatography and thin layerchromatography to give(R)—N′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylicacid tert-butyl ester (45.7 mg, yield 4.1%) as a white solid. R_(f)=0.39(1:3 EtOAc:n-Hexane); [α]²⁵ ₅₈₉ +26.25 (c 2.09, CH₃OH), ¹H NMR (400 MHz,CDCl₃) δ 7.15-7.03 (m, 3H), 6.07+6.00 (2d, 1H), 4.59+4.50 (2 dd, 1H),2.37+2.34 (2 s, 6H), 1.85-0.89 (m, 25H) ppm; HRMS (ESI) m/z calcd forC₂₂H₃₇N₂O₃ [M+H]⁺ 377.2804, found 377.2795, calcd for C₂₂H₃₆N₂NaO₃[M+Na]⁺ 399.2624, found 399.2618.

Example 50

Synthesis

Compound 1: 3-Methoxy-2-methyl-benzoic acid hydrazide was prepared usingmethodology described in US 2005/0209283 A1.

Compound 2: Pivaldehyde (13.45 g, 156 mmoles) was dissolved in 300 mLmethanol in a round bottom flask. 3-methoxy-2-methyl-benzoic acidhydrazide (26.6 g, 147.6 mmoles) and 150 drops glacial acetic acid wereadded, and the mixture was heated at reflux for ca. 8 hours, whilemonitoring by TLC. Upon completion of the reaction, the solvent wasremoved in vacuo, and the product was slurried in cold hexanes andfiltered on a Büchner funnel to provide 24.4 g (66%) of3-methoxy-2-methyl-benzoic acid (2,2-dimethyl-propylidene)-hydrazide.R_(f)=0.19, (2:1 hexanes:ethyl acetate); ¹H NMR (400 MHz, DMSO-d₆) δ7.42+7.41 (3 s, 1H), 7.1 (t, 1H), 6.9 (d, 1H), 6.82 (d, 1H), 3.78 (s,3H), 2.2+2.1 (2 s, 3H), 1.07+1.0 (3 s, 9H), multiple conformations,possibly E/Z mixture.

Compound 3:(S,S)-2-Allyl-2-chloro-3,4-dimethyl-5-phenyl-[1,3,2]oxazasilolidine wasprepared as described in Example 1.

Compound 4: 3-methoxy-2-methyl-benzoic acid(2,2-dimethyl-propylidene)-hydrazide (10.84 g, 43.31 mmol) was chargedto a dried, 1 L round bottom flask kept under a nitrogen atmosphere. 350mL anhydrous methylene chloride was added by catheter. The flask wascooled to about 5° C. in an ice bath.(S,S)-Allyl-2-chloro-3,4-dimethyl-5-phenyl-[1,3,2]oxazasilolidine (17.4g, 65 mmol) was added by syringe. Within minutes, the originally lightmixture turned a dark transparent yellow, and was left to stir undernitrogen at ca. 5° C. After 4 hr, TLC indicated a complete reaction. Thereaction was quenched by adding ca. 25 mL methanol with gentle mixing.The dark color dissipated. The solution was concentrated and the residuewas diluted with ethyl acetate (100 mL) and water (100 mL). The phaseswere separated and the aqueous layer was extracted with ethyl acetate(2×100 mL). The combined organic phases were washed once with brine,dried over MgSO₄, decanted, and concentrated to yield a deep yellow oilthat crystallized upon standing (13.66 g, 72.4% yield). The crudematerial was crystallized from 4:1 hexanes:ethyl acetate, whileseparating from an insoluble powdery residue, possibly pseudoephedrine.After one crystallization, the purified material indicated an e.e of ca.80%, favoring the R-isomer, as determined by Mosher analysis. After 3crystallizations, 6.65 g of (R)-3-methoxy-2-methyl-benzoic acidN′-(1-tert-butyl-but-3-enyl)-hydrazide (35.2%), was recovered as acrystalline solid, but without further improvement in ee R_(f)=0.38 (2:1hexanes:ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 7.22 (t, 1H), 7.11 (brs, 1H), 6.95 (d, 1H), 6.15 (m, 1H), 5.2 (d, 1H), 5.15 (d, 1H), 3.89 (s,3H), 2.80 (d, 1H), 2.50 (dm, 1H), 2.33 (s, 3H), 2.2 (dt, 1H), 1.08 (s,9H).

Compound 5 (title compound): (R)-3-methoxy-2-methyl-benzoic acidN′-(1-tert-butyl-but-3-enyl)-hydrazide (100 mg, 0.344 mmoles) and(R)-(+)-alpha-methyl-alpha-(trifluoromethyl)-phenylacetyl chloride (104mg, 0.413 mmoles) were dissolved in 1.5 mL methylene chloride in a vial.A solution of K₂CO₃ (95 mg, 0.69 mmoles) in ca. 0.5 mL water was added,and the reaction mixture was stirred at room temperature overnight, andmonitored by TLC. The phases were separated, adding additional methylenechloride and/or water as needed to aid manipulation. The methylenechloride layer was dried over MgSO₄ or Na₂SO₄, and solvent was removedin vacuo to provide crude product as an oily solid. This residue wastriturated with hexanes/ether, whereupon crystallization commenced.Crystallization was allowed to proceed without further disturbance.Isolation from the mother liquor provided 83 mg (16.4%) crystalline(R)-3-methoxy-2-methyl-benzoic acidN′-(1-tert-butyl-but-3-enyl)-N′-((S)-3,3,3-trifluoro-2-methoxy-2-phenyl-propionyl)-hydrazidefrom which was obtained an X-ray crystal structure. Mp=128-129° C.;[α]²⁵ ₅₈₉ +66.40 (c 2.00, CH₃OH); ¹H NMR (400 MHz, CDCl₃) δ 7.5 (d, 2H),7.2-7.3 (m, 4H), 7.07 (m, 1H), 6.9 (d, 1H), 6.6+6.2 (br, 1H), 5.2 (d,1H), 5.1 (d, 1H), 4.8 (1H, br), 3.85 (s, 3H), 3.8 (s, 3H), 2.55 (br,1H), 2.35 (br, 2H), 1.5 (3H), 1.05 (br s, 9H); ¹³C NMR (100 MHz, CDCl₃)δ 168.1, 158.3, 138.7, 134.0, 129.4, 128.0, 126.5, 126.4, 126.3, 124.9,122.1, 118.0, 117.0, 112.2, 57.3, 55.7, 35.8, 32.0, 28.1, 12.3; HRMS(ESI) m/z calcd for C₂₇H₃₄F₃N₂O₄ [M+H]⁺ 507.2471, found 507.2463, m/zcalcd for C₂₇H₃₃F₃N₂NaO₄ [+Na]⁺ 529.2290, found 529.2284. Anal. Cald forC₂₇H₃₃F₃N₂O₄: C, 64.02; H, 6.57; F, 11.25; N, 5.53; O, 12.63. Found: C,63.87; H, 6.71; N, 5.52.

In order to determine the stereochemical course of the asymmetricallylation reaction in Example 50, the x-ray crystal structure of(R)-3-methoxy-2-methyl-benzoic acidN′-(1-tert-butyl-but-3-enyl)-N′-((S)-3,3,3-trifluoro-2-methoxy-2-phenyl-propionyl)-hydrazidewas determined. This experiment established the absolute configurationof the carbon bearing the tert-butyl group and n-propyl group is R.

Example 51

Synthesis

Compound 1: (R)-3-methoxy-2-methyl-benzoic acidN′-(1-tert-butyl-but-3-enyl)-hydrazide was prepared as described inExample 50.

Compound 2: (R)-3-methoxy-2-methyl-benzoic acidN′-(1-tert-butyl-but-3-enyl)-hydrazide (3.05 g, 10.5 mmol) was dissolvedin 100 mL methanol. Palladium on charcoal (1%, 240 mg) was carefullyadded, and the mixture was shaken on a Parr hydrogenator for 2.5 hoursat a starting pressure of 55 psi. Decrease in pressure was monitored asa measure of the reaction progress; after 1.5 hours, hydrogen uptakeceased. The mixture was filtered through a pad of Celite, and solventwas removed in vacuo. Analysis by TLC using several solvents was largelyunsuccessful in distinguishing between the two hydrazide spots, although3:1 hexanes:ethyl acetate (R_(f)=0.28) was marginally successful. Thecrude product was crystallized twice from 2:1 pentane:hexanes toprovide >0.6 g of (R)-3-methoxy-2-methyl-benzoic acidN′-(1-tert-butyl-butyl)-hydrazide. ¹H NMR (400 MHz, CDCl₃) δ 7.05 (t,1H), 6.79 (d, 1H), 6.76 (d, 1H), 3.7 (s, 3H), 2.37 (m, 1H), 2.12 (s,3H), 1.7-1.1 (m, 4H), 0.90 (s, 9H), 0.82 (t, 3H).

Compound 3 (title compound): (R)-3-methoxy-2-methyl-benzoic acidN′-(1-tert-butyl-butyl)-hydrazide (100 mg, 0.34 mmol) and3,5-dimethoxy-4-methyl-benzoyl chloride (90 mg, 0.41 mmol) weredissolved in 1 mL methylene chloride. 1.5 eq. of ca. 25% K₂CO₃ wasadded, and the reaction mixture was stirred at room temperature andmonitored by TLC. Upon completion, the phases were separated, addingadditional CH₂Cl₂ and/or water as needed to aid manipulation. The CH₂Cl₂phase was dried over MgSO₄ or Na₂SO₄, and solvent was removed in vacuoto provide an oily solid. This was triturated with 1:1 hexane:ether andallowed to dry in air to yield (R)-3,5-Dimethoxy-4-methyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide.R_(f)=0.29 (2:1 hexanes:ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 7.1(t, 1H), 7.0 (s, NH 1H), 6.9 (d, 1H), 6.7 (s, 2H), 6.3 (d, 1H), 4.78 (m,1H), 3.95 (s, 3H), 3.85 (2 s, 6H), 2.2 (s, 3H), 2.05 (m, 1H), 1.95 (s,3H), 1.8 (m, 1H), 1.6 (m, 2H), 1.2+1.1+1.05 (3 s, 9H), 1.05 (m, 3H);HRMS (ESI) m/z calcd for C₂₇H₃₉N₂O₅ [M+H]⁺ 471.2859, found 471.2850,calcd for C₂₇H₃₈NaN₂O₅ [M+Na]⁺ 493.2670, found 493.2678.

Example 52

Synthesis

Compound 1: Benzyl carbazate is commercially available.

Compound 2: Benzyl carbazate (25 g, 150.44 mmoles) was dissolved in 200mL ethanol under a nitrogen atmosphere in a round bottom flask equippedwith magnetic stirring. Cyclohexanecarbaldehyde (17.7 g, 158 mmoles) andacetic acid (1 mL) were added to the solution and the mixture wasstirred at room temperature for 8 h. The resultant solid precipitate wasfiltered, washed with hexanes, crystallized from ethyl acetate, anddried overnight on the open bench to provide 28.87 g (73.7% yield) whitecrystalline N′-cyclohexylmethylene-hydrazinecarboxylic acid benzylester. R_(f)=0.67 (1:3 hexanes:ethyl acetate), ¹H NMR (400 MHz, CDCl₃) δ7.8 (1H, br s), 7.4 (5H, m), 7.1 (1H, br s), 5.3 (2H, s), 2.4 (1H, m),1.7-1.9 (4H, m), 1.3 (6H, m); HRMS (ESI) m/z calcd for C₁₅H₂₁N₂O₂ [M+H]⁺261.1603, found 261.1592, calcd for C₁₅H₂₀NaN₂O₂ [M+Na]⁺ 283.1426, found283.1417.

Compound 3:(S,S)-2-Allyl-2-chloro-3,4-dimethyl-5-phenyl-[1,3,2]oxazasilolidine wasprepared as described in Example 1.

Compound 4: A round bottom flask with magnetic stirrer and N₂ atmospherewas charged with 5 g (19.21 mmoles)N′-cyclohexylmethylene-hydrazinecarboxylic acid benzyl ester in 75 mLanhydrous CHCl₃. The mixture was chilled to 0° C., and(S,S)-oxazasilolidine (5.66 g, 21.1 mmoles) was added to the solutionusing a syringe. The ice bath was removed after ca. 30 minutes and thereaction was stirred at room temperature overnight. The reaction wasquenched with aq. Na₂CO₃. The organic layer was removed, and the aqueousphase extracted with CHCl₃. The combined organic phases were backwashedseveral times with water, dried over anhydrous Na₂SO₄, and freed ofsolvent using a rotary evaporator. The resultant crude product waspurified by column chromatography to provide(R)—N′-(1-cyclohexyl-but-3-enyl)-hydrazinecarboxylic acid benzyl esteras a light yellow viscous oil in 2.31 g, 39.8% yield. R_(f)=0.41 (3:1hexanes:ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 7.4 (5H, m), 6.3 (1H,br), 5.9 (1H, br s), 5.25 (2H, s), 5.2 (2H, br s), 2.95 (1H, br s), 2.35(1H, br d), 2.17 (1H, br m), 1.8 (2H, br m), 1.75 (2H, br d), 1.55 (1H,br t), 1.0-1.4 (6H, m) ppm; HRMS (ESI) m/z calcd for C₁₈H₂₇N₂O₂ [M+H]⁺303.2072, found 303.2079, calcd for C₁₈H₂₆NaN₂O₂ [M+Na]⁺ 325.1892, found325.1899.

Compound 5: R—N′-(1-cyclohexyl-but-3-enyl)-hydrazinecarboxylic acidbenzyl ester (2.20 g, 7.27 mmoles) was dissolved in 2 mL methylenechloride in a vial with a magnetic stirrer. Aqueous K₂CO₃ solution (1.51g, 10.91 mmoles in 4 mL) was added and the mixture was cooled on ice.The neat acid chloride was slowly added and ca. 1 mL methylene chloridewas used to chase and rinse. The mixture was stirred overnight, first onice then at room temperature, while monitoring by TLC. Water and/orCH₂Cl₂ was added to the reaction mixture to aid manipulation, and theorganic layer was collected. The aqueous layer was extracted once withCH₂Cl₂, and the organic layers were combined and dried over solidNa₂SO₄. Solvent was removed in vacuo, and the residue was trituratedwith 2:1 hexanes:ether to yield 2.93 g (92.7%) of(R)—N′-(1-cyclohexyl-but-3-enyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylicacid benzyl ester as a white solid. R_(f)=0.33 (3:1 hexanes:ethylacetate); ¹H NMR (400 MHz, DMSO-d₆) δ 9.8+9.65 (0.5H, NH, 2 s), 7.1-7.5(3.5H, m), 6.9-7.1 (5H, m), 6.05 (0.5H, m), 5.9 (0.5H, m), 5.2 (1H, m),5.0 (2H, m), 4.8 (1H, m), 4.35 (1H, m), 2.35 (2H, br m), 2.25 (6H, s),0.8-2.0 (11H, m); HMS (ESI) m/z calcd for C₂₇H₃₅N₂O₃ [M+H]⁺ 435.2647,found 435.2659, calcd for C₂₇H₃₄NaN₂O₃ [M+Na]⁺ 457.2467, found 457.2470.

Compound 6:(R)—N′-(1-Cyclohexyl-but-3-enyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic acid benzyl ester (2.91 g, 6.7 mmoles) was suspended in ca.50 mL glacial acetic acid. 102 mg 10% palladium on charcoal was added asa slurry in several mL of acetic acid. The mixture was diluted withacetic acid to 75 mL, and shaken on a Parr hydrogenator for 90 minutesat 20-30 psi. The suspension was allowed to stand for two days to allowthe carbon to settle out. The supernatant was filtered through a 0.45micron syringe filter into a recrystallizing dish and solvent wasevaporated to leave 2.06 g (102% mass balance) of(R)-3,5-dimethyl-benzoic acid N-(1-cyclohexyl-butyl)-hydrazide as areddish brown viscous oil. R_(f)=0.24 (2:1 hexanes:ethyl acetate); ¹HNMR (400 MHz, CDCl₃) δ 7.05 (1H, s), 7.0 (2H, s), 4.55+3.35 (1H, 2 t),2.4 (6H, s), 0.8-1.9 (15H, m), 0.9 (3H, t); HRMS (ESI) m/z calcd forC₁₉H₃₁N₂O [M+H]⁺ 303.2436, found 303.2441, calcd for C₁₉H₃₀N₂NaO [M+Na]⁺325.2256, found 325.2262.

Compound 7 (title compound): (R)-3,5-Dimethyl-benzoic acidN-(1-cyclohexyl-butyl)-hydrazide (0.14 g, 0.46 mmoles) was dissolved in1 mL of methylene chloride in a vial with a magnetic stirrer. AqueousK₂CO₃ solution (0.1 g, 0.69 mmoles, 2.77 mL) was added. The flask wascooled on ice, and neat acid chloride was added. Ca. 0.8 mL additionalmethylene chloride was used to chase and rinse. The mixture was stirredovernight, first on ice and then at room temperature, while monitoringthe reaction by TLC. Water and CH₂Cl₂ were added to the reaction mixtureto aid manipulation, and the aqueous layer was extracted once withCH₂Cl₂. The organic layers were combined and dried over solid Na₂SO₄.Solvent was removed in vacuo, and the crude product was purified bychromatography to yield 143 mg (67.9% yield) (R)-3,5-dimethyl-benzoicacid N-(1-cyclohexyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide asa reddish brown viscous oil. ¹H NMR (400 MHz, DMSO-d₆) δ 10.4 (1H, 2 s),7.0-7.25 (5H, m), 6.2 (1H, m), 4.45 (1H, m), 3.8 (3H, s), 2.35 (6H, s),2.0-2.2 (2H, m), 0.9-2.0 (15H, m), 0.9 (6H, m).

Examples 53 to 55 were prepared using methodology described in Example52. The percent enantiomeric excess (ee) was determined by chiral HPLC.

Example Structure % ee Name 53

62.4 (R)-3,5-Dimethyl- benzoic acid N′- benzoyl-N-(1- cyclohexyl-butyl)-hydrazide 54

59.1 (R)-3,5-Dimethyl- benzoic acid N-(1- cyclohexyl-butyl)-N′-(3-methoxy-benzoyl)- hydrazide 55

58.1 (R)-3,5-Dimethyl- benzoic acid N-(1- cyclohexyl-butyl)-N′-(4-ethyl-benzoyl)- hydrazide

Example 56

Synthesis

Compound 1: N′-Cyclohexylmethylene-hydrazinecarboxylic acid benzyl esterwas prepared as described in Example 52.

Compound 2:(R,R)-2-allyl-2-chloro-3,4-dimethyl-5-phenyl-[1,3,2]oxazasilolidine wasprepared using methodology described in Example 1 usingR,R-pseudephedrine.

Compound 3: (S)—N′-(1-cyclohexyl-but-3-enyl)-hydrazinecarboxylic acidbenzyl ester was prepared in 69% yield as a light yellow viscous oilusing methodology described in Example 52. Analytical data: R_(f)=0.41(3:1 hexanes:ethyl acetate); ¹H NMR (400 MHz, DMSO-d6) δ 8.65 (1H, brs), 7.4 (5H, m), 5.85 (1H, br), 5.1 (4H, m), 4.2 (1H, s), 2.7 (1H, brs), 2.1 (1H, br m), 2.0 (1H, br m), 1.8 (2H, br m), 1.65 (2H, br m),1.45 (1H, br m) 1.0-1.2 (6H, br m); ¹H NMR (400 MHz, CDCl₃) δ 7.4 (5H,m), 6.3 (1H, br), 5.9 (1H, br s), 5.25 (2H, s), 5.2 (2H, br s), 2.95(1H, br s), 2.35 (1H, br d), 2.17 (1H, br m), 1.8 (2H, br m), 1.75 (2H,br d), 1.55 (1H, br t), 1.0-1.4 (6H, m); HRMS (ESI) m/z calcd forC₁₈H₂₇N₂O₂ [M+H]⁺ 303.2072, found 303.2057, calcd for C₁₈H₂₆NaN₂O₂[M+Na]⁺ 325.1892, found 325.1873.

Compound 4:(S)—N′-(1-cyclohexyl-but-3-enyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic acid benzyl ester was prepared in 84.8% yield as a whitesolid using methodology described in Example 52. Analytical data:ee≧80%. R_(f)=0.34 (3:1 hexanes:ethyl acetate); ¹H NMR (400 MHz,DMSO-d₆) δ 9.8+9.65 (0.5H, NH, 2 s), 7.1-7.5 (3.5H, m), 6.9-7.1 (5H, m),6.05 (0.5H, m), 5.9 (0.5H, m), 5.2 (1H, m), 5.0 (2H, m), 4.8 (1H, m),4.35 (1H, m), 2.35 (2H, br m), 2.25 (6H, s), 0.8-2.0 (11H, m); HRMS(ESI) m/z calcd for C₂₇H₃₅N₂O₃ [M+H]⁺ 435.2647, found 435.2655, calcdfor C₂₇H₃₄NaN₂O₃ [M+Na]⁺ 457.2467, found 457.2469.

Compound 5: (S)-3,5-dimethyl-benzoic acidN-(1-cyclohexyl-butyl)-hydrazide was prepared using methodologydescribed in example 52. Analytical data: R_(f)=0.24 (2:1 hexanes:ethylacetate); ¹H NMR (400 MHz, DMSO-d₆) δ 7.05+6.95 (1H, s), 7.0+6.85 (2H, 2s), 4.55+4.05 (2H, 2 s), 4.3+3.15 (1H, 2 t), 2.3 (6H, 2 s), 0.6-1.9(15H, m), 0.9+0.8 (3H, 2 t); (400 MHz, CDCl₃) δ 7.05 (1H, s), 7.0 (2H,s), 4.55+3.35 (1H, 2 t), 2.4 (6H, s), 0.8-1.9 (15H, m), 0.9 (3H, t);HRMS (ESI) m/z calcd for C₁₉H₃₁N₂O [M+H]⁺ 303.2436, found 303.2440,calcd for C₁₉H₃₀N₂NaO [M+Na]⁺ 325.2256, found 325.2260.

Compound 6 (title compound): (S)-3,5-Dimethyl-benzoic acidN-(1-cyclohexyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide wasprepared using methodology described in Example 52 in 37% ee.

Examples 57 to 59 were prepared using methodology described in Example56. The percent enantiomeric excess (ee) was determined by chiral HPLC.Table 3 provides analytical data for examples 57 to 59.

Example Structure % ee name 57

87.7 (S)-3,5-Dimethyl- benzoic acid N′- benzoyl-N-(1- cyclohexyl-butyl)-hydrazide 58

86.7 (S)-3,5-Dimethyl- benzoic acid N-(1- cyclohexyl-butyl)-N′-(3-methoxy- bnezoyl)-hydrazide 59

99 (S)-3,5-Dimethyl- benzoic acid N-(1- cyclohexyl-butyl)- N′-(4-ethyl-benzoyl)-hydrazide

TABLE 3 Analytical Data Example ¹H NMR (solvent) MS 57 (CDCl₃) δ 8.0(1H, NH, br s), 7.0-7.8 (8H, [M + H⁺] m), 4.6 + 3.6 (1H, 2 br s),2.3-2.4 (6H, 2 br 407.2695 s), 1.4-2.0 (10H, br m), 1.1-1.4 (4H, 2 br[M + Na⁺] s), 0.9-1.1 (4H, 2 br s) ppm 429.2513 58 (CDCl₃) δ 6.9-7.6(7H + 1NH, m), [M + H⁺] 4.6 + 3.65 (1H, 2 br s), 3.85 (3H, 2 br s),437.2799 2.4 (6H, 2 br s), 1.4-2.0 (10H, br m), [M + Na⁺] 1.1-1.4 (4H,br m), 0.9-1.3 (4H, 2 br s) ppm 459.2616 59 (CDCl₃) δ 7.9 (1H, NH, brs), 6.9-7.6 (7H, [M + H⁺] m), 3.6 + 4.6 (1H, 2 br s), 2.75 (2H, br s),435.3016 2.4 (6H, 2 br s), 1.5-2.0 (9H, br m), [M + Na⁺] 1.1-1.4 (8H, brm), 1.0 (4H, 2 br s) ppm 457.2827

Example 60

Compound 1: 3-Methoxy-2-methyl-benzoic acid hydrazide was prepared asdescribed in Example 50.

Compound 2: 10.0 g (55.5 mmol) of 3-methoxy-2-methyl-benzoic acidhydrazide was dissolved in 200 mL of absolute ethyl alcohol.Cyclohexanecarbaldehyde (6.85 g, 61.0 mmol) and glacial acetic acid (3mL) were added, and the reaction mixture was stirred for 18 h whilemonitoring by TLC. The precipitate was collected by filtration andwashed with hexane. Product 3-methoxy-2-methyl-benzoic acidcyclohexylmethylene-hydrazide was obtained as a white solid (9.36 g,yield 61%): R_(f)=0.20 (3:1 EtOAc:n-Hexane); ¹H NMR (400 MHz, CDCl₃) δ11.17 (s, 1H), 7.91 (s, 1H), 7.50 (d, J=4.8 Hz, 1H), 7.25 (t, J=6.4 Hz,1H), 6.99 (t, J=5.6 Hz, 1H), 3.89 (s, 3H), 2.28 (s, 3H), 1.92-1.62 (m,5H), 1.38-1.14 (m, 6H); HRMS (ESI) m/z calcd for C₁₆H₂₃N₂O₂ [M+H]⁺275.1760, found 275.1752, calcd for C₁₆H₂₂N₂NaO₂ [M+Na]⁺ 297.1579, found297.1568.

Compound 3:(S,S)-2-Allyl-2-chloro-3,4-dimethyl-5-phenyl-[1,3,2]oxazasilolidine wasprepared as described in Example 1.

Compound 4: To a round bottom flask with a stirrer and a nitrogen inletwas added 3-methoxy-2-methyl-benzoic acid cyclohexylmethylene-hydrazide(2.8 g, 10.2 mmol) and dry CHCl₃ (50 mL). (S,S)-Oxazasilolidine (3.28 g,12.20 mmol) was added dropwise. The reaction was stirred at 50° C. for 6h, then at room temperature for 18 h. Saturated NaHCO₃ solution (30 mL)was added to quench the reaction. The CHCl₃ layer was separated and theaqueous layer was extracted with CHCl₃ (2×30 mL). The CHCl₃ solution wascombined and dried over anhydrous Na₂SO₄. The solvent was evaporated invacuo, and the crude mixture was purified by flash chromatography toobtain (R)-3-methoxy-2-methyl-benzoic acidN′-(1-cyclohexyl-but-3-enyl)-hydrazide as a white solid (1.74 g, 54%yield). ¹H NMR (400 MHz, CDCl₃) δ 9.69 (s, 1H), 7.24 (t, J=8.0 Hz, 1H),7.05 (d, J=8.0 Hz, 1H), 6.88 (d, J=7.6 Hz, 1H), 5.92 (m, 1H), 5.15 (dd,J=1.6, 17.2 Hz, 1H), 5.08 (dd, J=1.6, 10.4 Hz, 1H), 4.91 (dd, J=3.6, 6.8Hz, 1H), 3.83 (s, 3H), 2.75 (m, 1H), 2.26-2.22 (m, 1H), 2.16 (s, 3H),2.14-2.08 (m, 1H), 1.81-1.08 (m, 11H); HRMS (ESI) m/z calcd forC₁₉H₂₉N₂O₂ [M+H]⁺ 317.2229, found 317.2221, m/z calcd for C₁₉H₂₉N₂O₂[M+Na]⁺ C₁₉H₂N₂NaO₂ 339.2048, found 339.2037.

Compound 5 (title compound): To a solution of(R)-3-methoxy-2-methyl-benzoic acidN′-(1-cyclohexyl-but-3-enyl)-hydrazide (1.3 g, 4.11 mmol) in CH₂Cl₂ (5mL), was added K₂CO₃ (2.27 g, 16.44 mmol), H₂O (5 mL), and finally3,5-dimethylbenzoyl chloride (727 mg, 4.31 mmol). The reaction wasstirred at room temperature for 24 h. Additional CH₂Cl₂ and H₂O wereadded to aid manipulation, the CH₂Cl₂ layer was separated, and theaqueous layer was extracted with CH₂Cl₂. The CH₂Cl₂ solutions werecombined and dried over anhydrous Na₂SO₄. The solvent was evaporated invacuo and the residue was purified by flash chromatography to provide(R)-3,5-dimethyl-benzoic acidN-(1-cyclohexyl-but-3-enyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide asa white solid (1.45 g, 78% yield). R_(f)=0.21 (3:1 EtOAc:n-Hexane); ¹HNMR (400 MHz, DMSO-d₆) δ 10.47+10.39 (2 s, 1H), 7.20-7.01 (m, 5H), 6.37(d, J=7.2 Hz, 1H), 6.22-5.89 (m, 1H), 5.18 (dd, 1H), 5.02 (dd, 1H), 4.46(m, 1H), 3.79 (s, 3H), 2.44-2.34 (m, 2H), 2.29 (s, 3H), 2.02-1.53 (m,8H), 1.26-1.02 (m, 3H); HRMS (ESI) m/z calcd for C₂₈H₃₇N₂O₃ [M+H]⁺449.2804, found 449.2793, calcd for C₂₈H₃₆N₂NaO₃ [M+Na]⁺ 471.2624, found471.2615.

Example 61

Synthesis

(R)-4-Methoxy-3,5-dimethyl-benzoic acidN-(1-cyclohexyl-but-3-enyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazidewas prepared in 71% yield using methodology described in Example 60.Analytical data: R_(f)=0.14 (1:3 EtOAc:n-Hexane); ¹H NMR (400 MHz,DMSO-d₆) δ 10.46+10.38 (2 s, 1H), 7.21-6.42 (m, 5H), 6.16-5.93 (m, 1H),5.23 (dd, 1H), 5.01 (dd, 1H), 4.45 (m, 1H), 3.79 (s, 3H), 3.77 (s, 6H),2.42-2.28 (m, 2H), 2.04 (s, 3H), 1.88-1.05 (m, 14H); HRMS (ESI) m/zcalcd for C₂₉H₃₈N₂O₅ [M+H]⁺ 495.2859, found 495.2848, calcd forC₂₉H₃₈N₂NaO₅ [M+Na]⁺ 517.2678, found 517.2667.

Example 62

Synthesis

Compound 1: 4-ethyl-benzoic acid hydrazide is commercially available.

Compound 2: 4-ethyl-benzoic acid hydrazide (10 g, 60.9 mmoles) wasdissolved in 32 mL of methanol. Acetic acid (1 mL) was added, themixture was brought to reflux, and hydrocinnamaldehyde (5.77 g, 67mmoles) was then slowly added. The reaction mixture was refluxed for 10h and monitored by TLC. The precipitates were collected via filtrationand washed with hexanes. 4-Ethyl-benzoic acid(3-phenyl-propylidene)-hydrazide was obtained as a white solid (9.34 g,54.7% yield). R_(f)=0.36 (1:1 EtOAc:n-hexane); ¹H NMR (400 MHz, CDCl₃) δ8.87 (s, 1H), 7.72-7.24 (m, 10H), 2.87 (t, J=15.2 Hz, 2H), 2.79-2.57 (m,4H), 1.24 (t, J=8 Hz, 3H).

Compound 3: (R)-(+)-methyl p-tolyl sulfoxide (0.5 g, 3.21 mmoles) andtetrabutylammonium triphenyl-difluorosilicate (TBAT) (0.12 g, 0.214mmoles) were dissolved in 0.038 mL of 2-methyl-2-butene and 2.4 mL ofanhydrous methylene chloride under nitrogen and with magnetic stirring.Allyltrichlorosilane (0.23 mL, 1.61 mmoles) was added at −78° C. and thereaction was stirred for 15 minutes. 4-ethyl-benzoic acid(1-methyl-3-phenyl-propylidene)hydrazide (0.3 g, 1.07 mmoles) wasdissolved in 11.6 mL of anhydrous methylene chloride and added to thereaction over a period of 30 minutes. The reaction mixture was stirredfor 5 hours under nitrogen and maintained at −78° C. to −70° C., withTLC monitoring. The reaction was quenched with 1.5 mL triethylamine and3 mL anhydrous methanol at −78° C. Brine was then added and the mixturewas allowed to warm to room temperature. The organic layer was removed,combined with three methylene chloride extractions, and dried oversodium sulfate. The solvent was evaporated, and the crude mixture waspurified by flash chromatography using a step gradient of 2:1 and 1:1pentane:ethyl acetate. Purified (R)-4-ethyl-benzoic acidN′-(1-phenethyl-but-3-enyl)-hydrazide was obtained as a clear oil (0.145g, 41.4% yield, 57.6% ee by chiral HPLC on Chiralcel ADH). R_(f)=0.50(1:1 EtOAc:n-hexane); ¹H NMR (400 MHz, CDCl₃) δ 7.64-7.13 (m, 9H), 5.91(m, 1H), 5.15 (t, J=28.0 Hz, 2H), 3.12 (m, 1H), 2.76-2.63 (m, 4H),2.38-2.24 (m, 2H), 1.88-1.74 (m, 2H), 1.23 (t, J=8.0 Hz, 3H).

Compound 4 (title compound): (R)-4-ethyl-benzoic acidN′-(1-phenethyl-but-3-enyl)-hydrazide (0.1 g, 0.31 mmoles) was dissolvedin 0.47 mL of methylene chloride with magnetic stirring. Potassiumbicarbonate (0.064 g, 0.37 mmoles) in 0.7 mL of deionized water and3,5-dimethylbenzoyl chloride (0.063 g, 0.37 mmoles) were added to themixture. The reaction mixture was first stirred on ice and then allowedto warm to room temperature over the weekend while monitoring by TLC.The organic layer was removed and the aqueous layer was extracted withmethylene chloride three times and dried over sodium sulfate. Thesolvent was evaporated, and the crude mixture was purified by flashchromatography, using 2:1 hexanes:ethyl acetate as eluant. Purified(R)-3,5-dimethyl-benzoic acidN-(4-ethyl-benzoyl)-N-(1-phenethyl-but-3-enyl)-hydrazide was obtained asa yellow oil (0.102 g, yield 72.9%, 51.2% ee by chiral HPLC on ChiralcelADH). R_(f)=0.34 (2:1 hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃) δ 7.8-7.02(m, 12H), 5.99 (br, 1H), 5.22 (d, J=24.0 Hz, 2H), 2.73 (d, J=8 Hz, 2H),2.31 (s, 6H), 2.12-2.03 (m, 2H), 1.87 (s, 1H), 1.61 (s, 2H), 1.29 (t,J=8.0 Hz, 3H); HRMS (ESI) m/z calcd for C₃₀H₃₅N₂O₂ [M+H]⁺ 455.2699,found 455.2685, calcd for C₃₀H₃₄N₂NaO₂ [M+Na]⁺ 477.2518, found 477.2505.

Example 63

Synthesis

Compound 1: Compound 1 was prepared as described in Example 31.

Compound 2 (title compound): To a solution of(S)—N′-(1-tert-butyl-but-3-enyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylicacid benzyl ester (200 mg, 0.49 mmol) in THF (3 mL), was added boranedimethyl sulfide (2 M in THF, 125 μL, 0.25 mmol) at 0° C. The mixturewas stirred at room temperature for 16 h. Excess borane and THF wasremoved in vacuo, and additional THF (3 mL), followed by H₂O₂ (61microliters, 0.54 mmol) and 3N NaOH (90 microliters, 0.27 mmol) wereadded. After stirring at 50-55° C. for 1 h, more water was added, themixture was extracted with ether, and the ether layer was dried overanhydrous Na₂SO₄. The solvent was removed in vacuo and the residue waspurified by flash chromatography to give(S)—N′-(1-tert-butyl-4-hydroxy-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylicacid benzyl ester as a white solid (128 mg, yield 61%). R_(f)=0.30 (1:1EtOAc:n-hexane). ¹H NMR (400 MHz, DMSO-d₆) δ 9.71+9.63 (2 s, 1H),7.44-6.88 (m, 8H), 5.06 (dd, J=3.6, 12.8 Hz, 1H), 4.79 (dd, J=12.4, 25.2Hz, 1H), 4.43 (d, J=10.8 Hz, 1H), 4.28 (t, J=5.2 Hz, 1H), 3.47 (m, 2H),2.27 (s, 6H), 1.86-1.21 (m, 4H), 1.01 (s, 9H); HRMS (ESI) m/z calcd forC₂₅H₃₅N₂O₄ [M+H]⁺ 427.2597, found 427.2587, calcd for C₂₅H₃₄N₂NaO₄[M+Na]⁺ 449.2416, found 449.2407.

Example 64

Synthesis

(R)—N′-(1-tert-Butyl-4-hydroxy-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic acid benzyl ester was prepared in >99% ee using methodologydescribed in Example 63.

Example 65

This example illustrates the analytical method used to determine theenantiomeric excess (ee) of compounds of the invention.

As shown in FIG. 2A-C, the chiral HPCL chromatograms forrac-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide (Example 16),(R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide and(S)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide (Example 41)illustrates the method used to determine enantiomeric excess.rac-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide contains 50%of the R-isomer (peak 1 at approx. 16 minutes) and 50% of the S-isomer(peak 2 at approx. 19 minutes). The enantiomerically enriched sample of(R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide issubstantially free of the S-isomer with an ee of >99%. Theenantiomerically enriched sample of (S)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide issubstantially free of the R-isomer with an ee of >99%. In thisexperiment, data were generated using a 4.6 mm×25 cm Regis Rexchrom(S,S) ULMO column at a flow rate of 2.0 mL/min using 2% methanol inhexane as the solvent and an injection volume of 20 μL. The enantiomericexcess of other compounds of Formula II or III were determined insimilar fashion, unless otherwise noted.

Example 66

This example illustrates in vitro testing of compounds of the invention.

Gene Expression Cassette

GAL4 DBD (1-147)-CfEcR(DEF)/VP16AD-βRXREF-LmUSPEF: The wild-type D, E,and F domains from spruce budworm Choristoneura fumiferana EcR(“CfEcR-DEF”; SEQ ID NO: 1) were fused to a GAL4 DNA binding domain(“Gal4 DBD1-147”; SEQ ID NO: 2) and placed under the control of aphosphoglycerate kinase promoter (“PGK”; SEQ ID NO: 3). Helices 1through 8 of the EF domains from Homo sapiens RXRβ (“HsRXRβ-EF”;nucleotides 1-465 of SEQ ID NO: 4) and helices 9 through 12 of the EFdomains of Locusta migratoria Ultraspiracle Protein (“LmUSP-EF”;nucleotides 403-630 of SEQ ID NO: 5) were fused to the transactivationdomain from VP16 (“VP16AD”; SEQ ID NO: 6) and placed under the controlof an elongation factor-1α: promoter (“EF-1α”; SEQ ID NO: 7). Fiveconsensus GAL4 response element binding sites (“5XGAL4RE”; comprising 5copies of a GAL4RE comprising SEQ ID NO: 8) were fused to a syntheticTATA minimal promoter (SEQ ID NO: 9) and placed upstream of theluciferase reporter gene (SEQ ID NO: 10).

Stable Cell Line

CHO cells were transiently transfected with transcription cassettes forGAL4 DBD (1-147) CfEcR(DEF) and for VP16AD βRXREF-LmUSPEF controlled byubiquitously active cellular promoters (PGK and EF-1α, respectively) ona single plasmid. Stably transfected cells were selected by Zeocinresistance. Individually isolated CHO cell clones were transientlytransfected with a GAL4 RE-luciferase reporter (pFR Luc). 27-63 clonewas selected using Hygromycin.

Treatment with Chiral Diacylhydrazine Ligand

Cells were trypsinized and diluted to a concentration of 2.5×10⁴ cellsmL. 100 μL of cell suspension was placed in each well of a 96 well plateand incubated at 37° C. under 5% CO₂ for 24 h. Ligand stock solutionswere prepared in DMSO and diluted 300 fold for all treatments. Doseresponse testing consisted of 8 concentrations ranging from 33 μM to 0.0μM.

Reporter Gene Assay

Luciferase reporter gene expression was measured 48 h after celltreatment using Bright-Glo™ Luciferase Assay System from Promega(E2650). Luminescence was detected at room temperature using a Dynex MLXmicrotiter plate luminometer.

Stable Cell Line

A population of stably transformed cells containing CVBE and 6XEcRE asdescribed in Suhr et al., Proc. Natl. Acad. Sci. USA 95:7999-804 (1998)were obtained. Human 293 kidney cells, also referred to as HEK-293cells, were sequentially infected with retroviral vectors encoding firstthe switch construct CVBE, and subsequently the reporter construct6XEcRE Lac Z. The switch construct contained the coding sequence foramino acids 26-546 from Bombyx mori EcR (BE) (Iatrou) inserted in frameand downstream of the VP16 transactivation domain (VBE). A synthetic ATGstart codon was placed under the control of cytomegalovirus (CVBE)immediate early promoter and flanked by long terminal repeats (LTR). Thereporter construct contained six copies of the ecdysone response element(EcRE) binding site placed upstream of LacZ and flanked on both sideswith LTR sequences (6XEcRE).

Dilution cloning was used to isolate individual clones. Clones wereselected using 450 ug/mL G418 and 100 ng/mL puromycin. Individual cloneswere evaluated based on their response in the presence and absence oftest ligands. Clone Z3 was selected for screening and SAR purposes.

Human 293 kidney cells stably transformed with CVBE and 6XEcRE LacZ weremaintained in Minimum Essential Medium (Mediates, 10-010-CV) containing10% FBS (Life Technologies, 26140-087), 450 gum G418 (Mediates,30-234-CR), and 100 gnome promising (Sigma, P-7255), at 37° C. in anatmosphere containing 5% CO₂ and were subculture when they reached 75%confluence.

Treatment with Chiral Diacylhydrazine Ligand

Z3 cells were seeded into 96-well tissue culture plates at aconcentration of 2.5×10³ cells per well and incubated at 37° C. in 5%CO₂ for twenty-four hours. Stock solutions of ligands were prepared inDMSO. Ligand stock solutions were diluted 100 fold in media and 50 μL ofthis diluted ligand solution (33 μM) was added to cells. The finalconcentration of DMSO was maintained at 0.03% in both controls andtreatments.

Reporter Gene Assays

Reporter gene expression was evaluated 48 hours after treatment ofcells. β-galactosidase activity was measured using Gal Screen™bioluminescent reporter gene assay system from Tropix (GSY1000). Foldinduction activities were calculated by dividing relative light units(“RLU”) in ligand treated cells with RLU in DMSO treated cells.Luminescence was detected at room temperature using a Dynex MLXmicrotiter plate luminometer.

A schematic of switch construct CVBE, and the reporter construct 6XEcRELac Z is shown in FIG. 1. Flanking both constructs are long terminalrepeats, G418 and puromycin are selectable markers, CMV is thecytomegalovirus promoter, VBE is coding sequence for amino acids 26-546from Bombyx mori EcR inserted downstream of the VP16 transactivationdomain, 6× EcRE is six copies of the ecdysone response element, lacZencodes for the reporter enzyme β-galactosidase.

Gene Expression Cassette

GAL4 DBD-CfEcR(DEF)/VP16AD-MmRXRE: The wild-type D, E, and F domainsfrom spruce budworm Choristoneura fumiferana EcR (“CfEcR-DEF”; SEQ IDNO: 1) were fused to a GAL4 DNA binding domain (“Gal4 DBD1-147”; SEQ IDNO: 2) and placed under the control of the SV40e promoter of pM vector(PT3119-5, Clontech, Palo Alto, Calif.). The D and E domains from MusMusculus RXR (“MmRXR-DE”; SEQ ID NO: 11) were fused to thetransactivation domain from VP16 (“VP16AD”; SEQ ID NO: 6) and placedunder the control of the SV40e promoter of the pVP16 vector (PT3127-5,Clontech, Palo Alto, Calif.).

Stable Cell Line

CHO cells were transiently transfected with transcription cassettes forGAL4 DBD-CfEcR(DEF) and for VP16AD-MmRXRE controlled by SV40e promoters.Stably transfected cells were selected using Hygromycin. Individuallyisolated CHO cell clones were transiently transfected with a GAL4RE-luciferase reporter (pFR-Luc, Stratagene, La Jolla, Calif.). The 13B3clone was selected using Zeocin.

Treatment with Chiral Diacylhydrazine Ligand

Cells were trypsinized and diluted to a concentration of 2.5×10⁴ cellsmL. 100 μL of cell suspension was placed in each well of a 96 well plateand incubated at 37° C. under 5% CO₂ for 24 h. Ligand stock solutionswere prepared in DMSO and diluted 300 fold for all treatments. Doseresponse testing consisted of 8 concentrations ranging from 33 μM to0.01 μM.

Gene Switch Assay—Engineered EcR:USP/PXR systems

Cellular gene switch assays were performed by transiently transfectingthe following constructs in mouse embryonic fibroblast cells (NIH3T3).The wild-type D, E and F domains from Choristoneura fumiferana EcR orits V390I/Y410E/E274V mutant were fused to GAL4-DBD and placed under thecontrol of the CMV promoter in the pBIND vector (Promega Corporation,Madison, Wis., USA). The DEF domains of EcRs shown were amplified usingprimers designed based on 20-25 nt sequences at the 5′ and 3′ ends.Restriction enzyme sites EcoR I/BamH I and Xba I were added to 5′ and 3′primers respectively. The PCR products were digested with appropriaterestriction enzymes and cloned into pBIND vector. The EcR LBDs clonedinto pBIND vector were verified by sequencing. A chimeric RXR from Homosapiens RXRβ and Locusta migratoria RXR, fused to VP16-AD and placedunder the control of an SV40e promoter, was constructed as previouslydescribed (Kumar P & Katakam A in Gene Transfer, Friedmann T and RossiJ, eds, pp. 643-651 (2007), Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). The inducible luciferase reporter plasmid pFRLuc(Stratagene Cloning Systems, La Jolla, Calif., USA), contains fivecopies of the GAL4 response element and a synthetic minimal promoter.

NIH3T3 cells were maintained at 37° C. and 5% CO₂ in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% bovine calf serum, bothobtained from Mediatech Inc., Manassas, Va. Cells were planted in a96-well plate at a density of 2,500 cells/well in 50 μL of growthmedium. The following day cells were first treated with 35 μL ofserum-free DMEM containing dimethyl sulfoxide (DMSO; control) or a DMSOsolution containing ligand. Cells were then transfected with 15 μl ofserum-free DMEM containing 0.04 μg of EcR construct, 0.04 ug of RXRconstruct, and 0.16 μg of luciferase reporter construct per well, usingSuperFect™ transfection reagent (Qiagen Inc., Valencia, Calif., USA)according to the manufacturer's instructions. Ligands were tested at 8doses from 0.01-33 μM and the final DMSO concentration was 0.33% in bothcontrol and treatment wells. After a 48 hour post-treatment andtransfection incubation, the cells were assayed for luciferase activityusing the Bright-Glo™ Luciferase Assay System (Promega Corporation,Madison, Wis., USA) following the manufacturer's instructions. EC₅₀values were measured minimally in duplicate.

Reporter Gene Assay

Luciferase reporter gene expression was measured 48 h after celltreatment using Bright-Glo™ Luciferase Assay System from Promega(E2650). Luminescence was detected at room temperature using a Dynex MLXmicrotiter plate luminometer.

Each assay was conducted in two separate wells, and the two values wereaveraged. Fold inductions were calculated by dividing relative lightunits (“RLU”) in ligand treated cells with RLU in DMSO treated cells.EC₅₀s were calculated from dose response data using a three-parameterlogistic model. Relative Max FI was determined as the maximum foldinduction of the tested ligand (an embodiment of the invention) observedat any concentration relative to the maximum fold induction of GS™-Eligand (3,5-dimethyl-benzoic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide) observed at anyconcentration.

WT-CfEcR, 3T3 Cells rac R S Exam- EC₅₀ EC₅₀ EC₅₀ ple(s) A B R¹ R² (nM)RMFI^(a) (nM) RMFI (nM) RMFI 1.5^(b); PhCH₂O— 3,5-di-CH₃-Ph- CH₂═CHCH₂—(CH₃)₃C— >10,000 0.01 >10,000 0.00 31.4^(c) 48; 49 (CH₃)₃CO—3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— >33,000 0.02 15118 0.15 >33,000 0.032; 32 Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— ~100 0.60 ~30 0.74 ~3,3000.19 3; 33 2-CH₃-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 100 0.70 90.00.75 ~3,300 0.20 4 2-CH₃O-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— ~2,0000.32 993 0.38 5; 34 2-F-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 81 0.59~40 0.68 529 0.41 6; 35 2-Cl-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 590.72 ~20 0.88 ~1,000 0.44 7; 36 2-Br-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂—(CH₃)₃C— 86 0.63 78.7 0.58 ~3,300 0.11 8; 37 3-CH₃-Ph- 3,5-di-CH₃-Ph-CH₃CH₂CH₂— (CH₃)₃C— 193 0.88 ~30 0.86 ~3,300 0.22 9 3-CH₃O-Ph-3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 304 0.84 100.7 0.81 10 3-Cl-Ph-3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— ~100 0.50 94.5 0.87 11; 38 4-CH₃-Ph-3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 89 0.66 ~20 0.74 2168 0.25 12; 394-CH₃CH₂-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— ~20 0.79 23.6 0.78 4180.29 13 4-CH₃O-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— ~100 0.54 42.30.73 14 4-Cl-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— ~200 0.46 140 0.7415; 40 2,6-di-F-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 94 0.68 66.2 0.82~1,000 0.39 16; 41 2,6-di-Cl-Ph 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— ~3000.43 ~300 0.53 ~3,300 0.10 29; 47 2-CH₃-3- 3,5-di-CH₃-Ph- CH₃CH₂CH₂—(CH₃)₃C— 3.88 0.98 ~1 1.08 ~200 0.49 CH₃O-Ph- 1; 31 CH₃CH₂-3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 1.63 1.05 2.23 0.97 136 0.523-CH₃O-Ph- 17 3,4-di-CH₃O-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 41340.25 ~4,000 0.27 18 3,5-di-F-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 30.50.26 140.6 0.52 19; 42 3,5-di-CH₃O- 3,5-di-CH₃-Ph- CH₃CH₂CH₂—(CH₃)₃C— >10,000 0.00 ~5,000 0.09 >10,000 0.00 4-CH₃-Ph- 20 2-CH₃-3,4-3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 99.3 0.90 7.60 1.02 (OCH₂O)-Ph- 212-CH₃-3,4- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 1.32 0.86 0.304 0.87(OCH₂CH₂O)- Ph- 22 2-CH₃CH₂- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 0.4990.88 0.266 0.78 3,4-(OCH₂CH₂O)- Ph- 23 1-naphthyl- 3,5-di-CH₃-Ph-CH₃CH₂CH₂— (CH₃)₃C— 521 0.60 111 0.69 24 2-naphthyl- 3,5-di-CH₃-Ph-CH₃CH₂CH₂— (CH₃)₃C— ~500 0.44 ~200 0.46 25; 43 2-thienyl- 3,5-di-CH₃-Ph-CH₃CH₂CH₂— (CH₃)₃C— 3163 0.52 432 0.42 5385 0.13 26; 44 2,5-di-CH₃-3-3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 2145 0.76 ~1,000 1.16 ~10,000 0.07furanyl- 27; 45 2-C1-3- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 95.2 0.707059 0.32 pyridyl- 28; 46 6-Cl-3- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— (CH₃)₃C— 4700.70 243 0.38 ~20,000 0.05 pyridyl- 51 2-CH₃-3- 3,5-di-CH₃O-4-CH₃CH₂CH₂— (CH₃)₃C— 80.2 0.82 ~70 1.00 CH₃O-Ph- CH₃-Ph- 60 2-CH₃-3-3,5-di-CH₃-Ph- CH₂═CHCH₂— cyclohexyl ~1000 0.49 ~2,000 0.62 CH₃O-Ph- 612-CH₃-3- 3,5-di-CH₃O-4- CH₂═CHCH₂— cyclohexyl ~3,300 0.31 ~3,300 0.33CH₃O-Ph- CH₃-Ph- 53; 57 Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— cyclohexyl ~3,3000.30 2755 0.32 54; 58 3-CH₃O-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂— cyclohexyl~3,300 0.19 ~3,300 0.25 55; 59 4-CH₃CH₂-Ph- 3,5-di-CH₃-Ph- CH₃CH₂CH₂—cyclohexyl ~2,000 0.26 ~1,000 0.28 52; 56 2-CH₃CH₂- 3,5-di-CH₃-Ph-CH₃CH₂CH₂— cyclohexyl 220 0.38 159 0.37 3-CH₃O-Ph- 62 4-CH₃CH₂-Ph-3,5-di-CH₃-Ph- CH₂═CHCH₂— PhCH₂CH₂— ~5,000 0.06 3093 0.14 63; 64 PhCH₂O—3,5-di-CH₃-Ph- HO(CH₂)₃C— (CH₃)₃C— >33,000 0.02 >10,000 0.00 ^(a)RMFI =relative maximum fold induction; ^(b)“1.5” denotes Compound 5 of Example1; ^(c)“31.4” denotes Compound 4 of Example 31.

[V390I/Y410E/E274V]-CfEcR, 3T3 Cells rac R S Example(s) A B R¹ R² EC₅₀(nM) RMFI^(a) EC₅₀ (nM) RMFI EC₅₀ (nM) RMFI  1.5^(b); 31.4^(c) PhCH₂O—3,5-di-CH₃- CH₂═CHCH₂— (CH₃)₃C— ~2000 0.36 >10,000 0.00 Ph- 48; 49(CH₃)₃CO— 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— ~4000 0.50 2749 0.59 6605 0.20Ph-  2; 32 Ph- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 3.9 0.85 2.2 0.91 2670.38 Ph-  3; 33 2-CH₃-Ph- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 12.2 0.97 1.10.69 ~400 0.48 Ph-  4 2-CH₃O- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— ~300 0.7299 0.52 Ph- Ph-  5; 34 2-F-Ph- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 1.1 0.67~0.8 1.16 86 0.64 Ph-  6; 35 2-Cl-Ph- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C—1.2 0.95 0.63 1.01 ~20 0.77 Ph-  7; 36 2-Br-Ph- 3,5-di-CH₃- CH₃CH₂CH₂—(CH₃)₃C— ~3 1.28 1.4 0.90 433 0.36 Ph-  8; 37 3-CH₃-Ph- 3,5-di-CH₃-CH₃CH₂CH₂— (CH₃)₃C— 16.2 0.86 1.2 0.92 292 0.47 Ph-  9 3-CH₃O-3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 7.1 0.63 1.4 0.70 Ph- Ph- 10 3-Cl-Ph-3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 23.9 0.86 ~3 0.76 Ph- 11; 38 4-CH₃-Ph-3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 6.3 0.73 ~1 0.90 341 0.44 Ph- 12; 39 4-3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— ~0.6 0.96 0.56 0.82 ~20 0.70 CH₃CH₂- Ph-Ph- 13 4-CH₃O- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— ~5 0.71 1.4 0.68 Ph- Ph-14 4-Cl-Ph- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— ~20 0.83 51 0.74 Ph- 15; 402,6-di-F- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— ~1 0.92 0.54 0.79 ~20 0.69 Ph-Ph- 16; 41 2,6-di-Cl- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— ~30 0.73 51.2 0.83~700 0.62 Ph- Ph- 29; 47 2-CH₃-3- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 0.150.77 0.078 1.05 56.3 0.57 CH₃O-Ph- Ph-  1; 31 2- 3,5-di-CH₃- CH₃CH₂CH₂—(CH₃)₃C— 0.14 1.05 0.13 0.82 ~30 0.57 CH₃CH₂- Ph- 3-CH₃O- Ph- 17 3,4-di-3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 720 0.51 ~300 0.53 CH₃O-Ph- Ph- 183,5-di-F- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— ~3 1.00 ~10 0.79 Ph- Ph- 19;42 3,5-di- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 3928 0.15 ~2000 0.37 ~70000.08 CH₃O-4- Ph- CH₃-Ph- 20 2-CH₃- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 12.10.92 0.26 1.01 3,4- Ph- (OCH₂O)- Ph- 21 2-CH₃- 3,5-di-CH₃- CH₃CH₂CH₂—(CH₃)₃C— ~0.1 0.85 ~0.04 1.14 3,4- Ph- (OCH₂CH₂O)- Ph- 22 2- 3,5-di-CH₃-CH₃CH₂CH₂— (CH₃)₃C— 0.059 0.79 0.064 0.77 CH₃CH₂- Ph- 3,4- (OCH₂CH₂O)-Ph- 23 1- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 66 0.84 ~10 0.73 naphthyl- Ph-24 2- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 61 0.62 ~20 0.80 naphthyl- Ph- 25;43 2-thienyl- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 172 0.89 ~20 0.72 10650.52 Ph- 26; 44 2,5-di- 3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 84 0.97 99.40.95 ~2000 0.49 CH₃-3- Ph- furanyl- 27; 45 2-Cl-3- 3,5-di-CH₃-CH₃CH₂CH₂— (CH₃)₃C— 7.0 0.73 1469 0.80 pyridyl- Ph- 28; 46 6-Cl-3-3,5-di-CH₃- CH₃CH₂CH₂— (CH₃)₃C— 47 0.68 ~20 0.78 3995 0.42 pyridyl- Ph-51 2-CH₃-3- 3,5-di- CH₃CH₂CH₂— (CH₃)₃C— 15 0.81 0.56 0.81 CH₃O-Ph-CH₃O-4- CH₃-Ph- 60 2-CH₃-3- 3,5-di-CH₃- CH₂═CHCH₂— cyclohexyl ~200 0.73746 0.78 CH₃O-Ph- Ph- 61 2-CH₃-3- 3,5-di- CH₂═CHCH₂— cyclohexyl 521 0.391016 0.67 CH₃O-Ph- CH₃O-4- CH₃-Ph- 53; 57 Ph- 3,5-di-CH₃- CH₃CH₂CH₂—cyclohexyl 629 0.62 769 0.50 Ph- 54; 58 3-CH₃O- 3,5-di-CH₃- CH₃CH₂CH₂—cyclohexyl 474 0.44 121 0.42 Ph- Ph- 55; 59 4- 3,5-di-CH₃- CH₃CH₂CH₂—cyclohexyl ~70 0.42 ~30 0.35 CH₃CH₂- Ph- Ph- 52; 56 2- 3,5-di-CH₃-CH₃CH₂CH₂— cyclohexyl ~200 0.64 107 0.52 CH₃CH₂- Ph- 3-CH₃O- Ph- 62 4-3,5-di-CH₃- CH₂═CHCH₂— PhCH₂CH₂— 2106 0.67 1991 0.54 CH₃CH₂- Ph- Ph- 63;64 PhCH₂O— 3,5-di-CH₃- HO(CH₂)₃— (CH₃)₃C— ~8000 0.33 >10,000 0.00 Ph-^(a)RMFI = relative maximum fold induction; ^(b)“1.5” denotes Compound 5of Example 1; ^(c)“31.4” denotes Compound 4 of Example 31.

Example 67

This example illustrates in vivo testing of compounds of the invention.In vivo induction of a reporter enzyme with chiral diacylhydrazineligands of the present invention was evaluated in a C57BL/6 mouse modelsystem containing a gene switch.

Gene Expression Cassettes

The wild-type D, E, and F domains from spruce budworm Choristoneurafumiferana EcR (“CfEcR-DEF”; SEQ ID NO: 1) were mutated [V107 (gtt)→I107(att) and Y127 (tac)→E127 (gag)] and fused to a GAL4 DNA binding domain(“Gal4 DBD1-147”; SEQ ID NO: 2). Helices 1 through 8 of the EF domainsfrom Homo sapiens RXRβ (“HsRXRβ-EF”; nucleotides 1-465 of SEQ ID NO: 4)and helices 9 through 12 of the EF domains of Locusta migratoriaUltraspiracle Protein (“LmUSP-EF”; nucleotides 403-630 of SEQ ID NO: 5)were fused to the transactivation domain from VP16 (“VP16AD”; SEQ ID NO:6), which regulates a reporter gene human secreted alkaline phosphatase(“SEAP”, SEQ ID NO: 12) that was placed under the control of a 6×GAL4response element (SEQ ID NO: 13) and a transthyretin promoter (SEQ IDNO: 14). Each element of the gene switch was on a separate plasmid.Receptor expression was under the control of a CMV promoter (SEQ ID NO:15). Induction was evaluated by the amount of reporter protein expressedin the presence of ligand.

Electroporation of Gene Switch

SEAP expression in serum of mice was evaluated after electroporation ofthe gene switch into mouse quadriceps. Mice were anesthetized with 2μL/g of a mixture of ketamine (100 mg/mL) and xylazine (20 mg/mL).Animals were then shaved, DNA vectors injected into the muscle in avolume of 2×50 μL polyglutamic acid (12 mg/mL water), electrodeconductivity gel applied, and an electrode caliber (1 cm×1 cm; model384) was placed on hind leg. The muscle was electroporated with 200V/cm, 8 times, for 20 msec/pulse, at 1 sec time intervals. Thetransverse electrical field direction was reversed after the animalsreceived half of the pulses. Electroporation was performed with an ECM830 electroporator from BTX Molecular Delivery Systems.

Treatment with Chiral Diacylhydrazine Ligand

In some experiments mice received an intraperitoneal injection (IP) of2.6 μmol of ligand in 50 μL of DMSO 3 days after electroporation of thegene switch. In other experiments the concentration of ligand wasdecreased to 26 nmol/50 μL of DMSO/mouse. SEAP expression was evaluated2-11 days after ligand administration. In other experiments ligand wasadministered in rodent chow. The chow was prepared by dissolving 2 g ofligand in 20 mL of acetone and adding it to 1 kg of LabDiet 5010autoclavable chow from Purina Mills. This was thoroughly mixed in aHobart mixer and then mixed for an additional 15 min in a Cross Blendmixer. Animals received chow ad libitum for 1, 2, or 3 days. All valuesare the average from four animals. Background SEAP in sera from animalstreated with vector alone without ligand addition was 0-11 ng/mL serum.

Reporter Assay

Mouse serum was obtained by centrifugation of blood acquired byretroorbital bleeding with a small glass capillary tube. SEAPquantification was determined using a Clontech Great Escapechemiluminescence kit and by comparison with the Clontech SEAP standard.

To evaluate the relative in vivo effectiveness of(R)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide (Example 1)to induce RheoSwitch® Therapeutic System (RTS) gene expression, twelveC57BL6 mice were electroporated with the following plasmids:pCMV/GEVY(DEF), pCMV/VP16-Hs-LmRXR and p6XGAL4RE-TTR-SEAP. The plasmidpCMV/GEVY(DEF) consists of the D, E and F domains from Choristoneurafumiferana EcR carrying the mutations V390I/Y410E/E274V fused downstreamof the yeast GAL4-DBD (aa 1-147) and placed under the control of the CMVpromoter and a downstream SV40 polyadenylation signal in the pBINDvector (Promega Corporation, Madison, Wis., USA). The DEF domains ofEcRs shown were amplified using primers designed based on 20-25 ntsequences at the 5′ and 3′ ends. Restriction enzyme sites BamH I and XbaI were added to 5′ and 3′ primers respectively. The PCR products weredigested with appropriate restriction enzymes and cloned into pBINDvector (FIG. 20). The vector pCMV/VP16-Hs-LmRXR contains a chimeric RXRfrom Homo sapiens RXRβ (helix 1-8 of E domain) and Locusta migratoriaRXR (helix 9-12 of E domain), fused downstream of VP16-activation domainand placed under the control of the CMV promoter in the pBIND vector(FIG. 21). The inducible SEAP reporter vector p6xGAL4RE-TTR-SEAPcontains the human secreted alkaline phosphatase reporter gene placedunder the control of the inducible promoter consisting of 6 copies ofthe Gal4 response element upstream of the transthyretin (TTR) promoter(FIG. 22). Three days after electroporation of plasmid DNA into thequadriceps of mice, the three ligand preparations were administered tomice by intraperitoneal injection at a rate of 26 nmol/mouse (11.4μg/mouse, approximately 570 μg/kg). Human secreted alkaline phosphatase(SEAP) expression in mouse serum was subsequently monitored forseventeen days after ligand administration. The relative in vivoeffectiveness of (S)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide (Example 31)and rac-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide wasevaluated for comparison. As shown in FIG. 3,(S)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide (●-●) doesnot induce RTS gene reporter expression. rac-2-Ethyl-3-methoxy-benzoicacid N′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide inducesmodest RTS gene reporter expression (▴-▴). (R)-2-Ethyl-3-methoxy-benzoicacid N′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide (◯-◯)induces substantial RTS gene reporter expression.

Example 68 Physical Properties of the R Enantiomer and Racemate of2-Ethyl-3-methoxy-benzoic AcidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide

Morphic Form: The solid sample lots are reported in FIG. 4. With respectto the R enantiomer, samples obtained by rapidcrystallization/precipitation from either methanol/water ortoluene/heptane yield the same powder X-ray diffraction pattern (datanot shown), and have essentially the same melting point([toluene/heptane]166.2-167.1° C., [CH₃OH/H₂O]166.5-167.4° C.) ascompared to each other and as compared to a standard obtained from CH₃OHcrystallization (165.1-166.5° C.). With respect to the racemate, twoseparate preparations obtained by methanol evaporation gave the samemelting point (170-171° C., 169-170° C.) within experimental error andslight variations in purity. The morphic forms of the racemate and Renantiomer of 2-Ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide appear to bethe most typical.

Micronization and Particle Size: To normalize for particle size, each ofthe R enantiomer and racemate of 2-Ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide werepre-screened through 20 mesh and micronized. Particle size wasdetermined by weighing out about 20 mg of the test substance into a 15mL Falcon tube. 10 mL of deionized water were added into the powder. Thesuspension was mixed, sonicated for 20 minutes, and then immediatelyanalyzed for particle size by laser light diffraction using a MalvernMastersizer device. The size distributions appear in FIGS. 5 and 6.Although the racemate appears to have a slightly larger overall particlesize than the R enantiomer, the two samples are quite similar.

Bulk and tapped density analysis: Bulk densities of micronized Renantiomer and racemate of 2-Ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide werecalculated using the measured weight and volume in a 10 mL a graduatedcylinder. Tapped density of micronized material was calculated using thematerial weight and tapped volume. The tapped volume was measured usingtap density analyzer (model: Stampfvolumeter, STAV2003 by JEL), thetapped volume was recorded when it became constant (FIG. 7). The Renantiomer has a slightly lower bulk density and tapped density. Bothmaterials are flocculent (low bulk density).

Thermal gravimetric analysis/Differential Thermal Analysis (TGA/DTA)Analysis: Micronized R enantiomer and racemate of2-Ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide wereanalyzed by thermal gravimetric analysis/differential thermal analysis.The thermal gravimetry plot appears in FIG. 8. Both materials showed anendothermal event on the DTA profile. The onset temperature for the Renantiomer (163.6° C.) was significantly lower than that of the racemate(171.2° C.). Heat of fusion for the R enantiomer (59.8 uv·s/mg) was alsosignificantly lower than that of the racemate (80.8 uv·s/mg). Thisillustrates the two materials are different crystalline forms. The lowermelting point and lower heat of fusion of the R enantiomer are reflectedin differing solubility properties.

Upon heating to above 150° C., R enantiomer lost 0.4% of total weightand the racemate lost 0.8% of total weight (FIG. 8). The moisturecontent (heat-removable moisture) in the two materials is slightlydifferent.

Dynamic Vapor Sorption Analysis: When exposed to a desiccant condition(20% RH), the racemate did not show any significant weight loss, whereasthe R enantiomer lost 0.9% of total weight (data not shown) which maycorrespond to loosely associated moisture acquired during sampletransfer or storage. After dehydration under dessicant conditions, thetwo materials showed comparable weight gains upon exposing to low(30-40% RH), medium (50-60% RH) and high (70-80% RH) humidity. At 70%RH, the weight gain is 6.3% for the R enantiomer and 6.2% for theracemate (data not shown), indicating moderate hygroscopicity for bothmaterials.

Solubility in Pharmaceutical Excipients: Assay 1: The solubility ofmicronized R enantiomer and racemate of 2-Ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide was assessedqualitatively in commercially-available excipients (FIG. 9). Initialsolutions/suspensions of 10 mg/g (test article/excipient) in glass vialswere prepared by sonication at room temperature for 15 minutes withswirling for 30 seconds at 5 minute intervals, followed by heating in analuminum block at 50° C. for 12 minutes with swirling for 30 seconds at3 minute intervals. Based on the appearance of this initial 10 mg/gexcipient sample, a 2-4 point concentration continuum was scanned by upor down sampling at several of the following points: 5, 10, 15, 20, 30,50 mg/g (test article/excipient). The R enantiomer was more soluble thanracemate in all the tested pharmaceutical excipients except for neatpolysorbate 80, in which the R enantiomer and the racemate appearcomparably soluble (see FIG. 9). No change in color was observed for anyof the samples. Photos of several of the preparations in clear glassvials were taken (not shown).

Assay 2: For each excipient, the lowest concentration at which Renantiomer or racemate of 2-Ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide is insolublein the FIG. 9 assay was identified. Suspensions of both materials atthis same concentration were prepared in vials. Each sample was stirredat room temperature for ≦2.5 hr (treatment 1). As a separate experiment,test article/excipient combinations were heated in an aluminum block at90° C. for 5 minutes, or longer as needed; indicated in FIG. 10, andallow to cool to room temperature, and seeded with a tiny amount of thesame micronized substance. (treatment 2). The physical appearance wasrecorded at 72 hr or the indicated time (FIG. 10). In all cases, nochange in color was observed. This solubility assay operates from afully dissolved condition in which initial particle size and morphicform are irrelevant. The observation of precipitation/crystallizationupon seeding supersaturated 2-Ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide racematesolutions with any morphic form of crystalline racemate, while Renantiomer remains fully dissolved (especially without heating)indicates a solubility differential between the racemate and the Renantiomer at the tested concentration. Photos were taken ofpreparations in clear glass vials (R enantiomer under the conditions ofassay 1 without heating to 90° C., and racemate after 90° C. treatment,cooling and seeding, not shown).

Assay 3: R enantiomer and racemate of 2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide were mixedwith 20% PEG 1000 in distilled water, pH 7.0 at 1-2 mg of powder per mLThe mixture was incubated at 37° C., vortexed at room temperature forca. 2 hours, and allowed to stand for another 12 hours at roomtemperature. The solution was filtered or centrifuged, andconcentrations were quantified by duplicate LC/MS/MS injections, using ageneric LC/MS/MS method with a minimum 4 point calibration curve (FIG.11). See Löbenberg R, et al., Solubility as a limiting factor to drugabsorption, In: Oral Drug Absorption, J. B. Dressman (Ed), MarcelDekker, NY, 2000 and Mader W J, Grady L T, Determination of solubility,Chapter 5 in Techniques of Chemistry, Physical Methods in Chemistry,Vol. 1, Part V, Weissberger, V. A., Rossiter, B. W., Eds., Wiley, NewYork, 1971.

Assay 4: Equilibrium solubility in formulation excipients and biologicalfluids influences drug bioavailability, although kinetics of dissolutionmay also be a limiting factor in determining bioavailability. Thesolubilities of R enantiomer and racemate of 2-ethyl-3-methoxy-benzoicacid N′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide wereestablished quantitatively in aqueous preparations of the pharmaceuticalsurfactant polysorbate 80 and then the intrinsic dissolution rate ofcompacted test substance was observed. Mixtures of racemate and Renantiomer in 0.5%, 1%, and 1.5% polysorbate 80 solutions were preparedby addition of 20+/−1 mg test substance in a 2 mL Eppendorf vial,followed by 500 mg polysorbate solution. The mixture was bead-beaten for120 second, shaken at 25° C. for 16 to 24 hours, and filtered using aSpin-X (0.2 μM). The pH of the filtrate was recorded. The concentrationof test substance in the filtrate was measured by quantitative HPLCanalysis (FIG. 12). The R enantiomer is 2-3× more soluble in 1.0 and1.5% polysorbate 80 solutions. In deionized water and 0.5% polysorbate80, the solubility of racemate and R enantiomer are comparable.

Next, the intrinsic dissolution profiles of R enantiomer and racemate of2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide weredetermined using compressed pellets (7 mm diameter, 100 mg) in 1%Polysorbate 80 in Di-water at 37° C., according to the following testconditions:

Vessel: 1000 mL

Medium: 1% Polysorbate 80 in di-water (vacuum filtered thru a 0.8 μMfilter)

Medium volume: 1000 mL

Speed: 100 RPM

Temp: 37° C.

Sampling points: 15, 30, 45, 60, 90 and 120 min

Sampling volume: 1 mL (filtered thru 30 micron)

Analysis: HPLC (prepare standard solutions from each lot)

Sample size: n=2 pellets/test substance

The release of racemate and R enantiomer was under the detection limitby HPLC up to 120 minutes. After 120 minutes, it was observed that thepellets for both substances remained intact in the medium.

Example 69 Cellular Membrane Permeability Properties of the R Enantiomerand Racemate of 2-Ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide

Bidirectional Permeability Through Caco-2 Cell Monolayers (see Sambuy Y,et al., The Caco-2 cell line as a model of the intestinal barrier:influence of cell and culture-related factors on Caco-2 cell functionalcharacteristics. Cell Biol Toxicol. 2005 January; 21(1):1-26 andArtursson P, et al., Caco-2 monolayers in experimental and theoreticalpredictions of drug transport. Adv Drug Deliv Rev. 2001 Mar. 1;46(1-3):27-43):

Data is shown in FIG. 13. The test compounds were prepared as 5 μM inHBSSg with a maximum DMSO concentration ≦1%. Confluent monolayers ofCaco-2 cells, 21 to 28 days old were prepared. The receiver well wasprepared with 1% BSA in modified Hanks buffer HBSSg, and the apical andbasolateral sides of Caco-2 cell were maintained at pH 7.4±0.2. Twomonolayers were dosed in each direction (N=2): the apical side was dosedfor (A→B) assessment and the basolateral side was dosed for (B→A)assessment. The test compound concentration was measured for both apicaland basolateral sides at a time point of 120 minutes using a genericLC/MS/MS method with a minimum 4 point calibration curve. The values inFIG. 13 are: the percent recovery of the test compound from Transwell®wells containing Caco-2 cell monolayers; apparent permeability (Papp) inboth directions; efflux ratio (Papp B→A)/(Papp A→B); absorptionpotential of a test compound classified as either Low or High; effluxclassification is significant when efflux ≧3.0 and Papp (B→A)≧1.0×10-6cm/s. The R enantiomer is more permeable than the racemate in bothdirections. Also, the efflux rate for the R enantiomer is lower.

Blood Brain Barrier Penetration Potential Determination Using MDR1-MDCKCells (see Taub M E, et al., Functional assessment of multipleP-glycoprotein (P-gp) probe substrates: Influence of cell line andmodulator concentration on P-gp activity. Drug Metab Dispos. 2005November; 33(11):1679-87 and Wang Q, et al., Evaluation of the MDR-MDCKcell line as a permeability screen for the blood-brain barrier. Int JPharm. 2005 Jan. 20; 288(2):349-59. (revised from Absorption SystemsInc. bulletin):

Data is shown in FIG. 14. The test compounds were prepared as a 5 μMsolution in HBSSg with a maximum DMSO concentration ≦1%. Confluentmonolayers of MDR1-MDCK cells, 7 to 11 days old were prepared. Thereceiver well was prepared with 1% BSA in modified Hanks buffer (HBSSg),and the apical and basolateral sides were maintained at pH 7.4±0.2. Twomonolayers were dosed in each direction (N=2): the apical side was dosedfor (A→B) assessment and the basolateral side was dosed for (B→A)assessment. Test compound concentration was measured for both apical andbasolateral sides at a time point of 120 minutes using a genericLC/MS/MS method with a minimum 4 point calibration curve. The reportedvalues in FIG. 14: the percent recovery of the test compound from theTranswell® wells containing MDR1-MDCK cell monolayers; apparentpermeability (Papp) in both directions; efflux ratio (Papp B→A)/(PappA→B); and the blood-brain barrier penetration potential classification.R enantiomer of 2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide is morepermeable than racemate in both A→B and B→A directions in MDR1-MDCKcells. Also, R enantiomer experiences less efflux. The R enantiomer isclassified as moderately penetrating through the blood-brain barrier,whereas reacemate is classified as low. Higher blood-brain barrierpenetration is a significant advantage for medical indications wherecentral nervous system (CNS) penetration is required.

Example 70 Microsomal Metabolism of the R Enantiomer and Racemate of2-Ethyl-3-methoxy-benzoic AcidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide (see JeffreyP, et al., Utility of Metabolic Stability Screening: Comparison of InVitro and In Vivo Clearance. Xenobiotica. 2001 August-September;31(8-9):591-8 and Lin J H, et al., Role of Pharmacokinetics andMetabolism in Drug Discovery and Development. Pharmacol Rev. 1997December; 49(4):403-49)

The test compounds were prepared as 5 μM solutions in DMSO(concentration less than 0.25%), methanol, or acetonitrile. Mixed genderhuman liver microsomes were pooled from ≧10 donors, and incubated with 1mM NADPH. The test compound was incubated at 37° C. in buffer containing1.0 mg/mL of microsomal protein with 1 mM NADPH. At 0 and 60 minutes,the mixture was sampled and monitored by LC/MS/MS for the peak areacorresponding to the test compound, with the range of 10-100%. The assaywas run in duplicate (N=2 separate incubations). The data reported inFIG. 15 is percent remaining of testosterone standard and percentremaining of the test compound. R enantiomer is slightly more stable tometabolism in human liver microsomes than is the racemate.

Example 71 Bioavailability of the R Enantiomer and Racemate of2-Ethyl-3-methoxy-benzoic AcidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide

Compounds in Labrasol were administered to C57BL/6N:Crl (wild-type) mice(Charles River Laboratories) by oral gavage, the intended route ofadministration in humans. Dosage was set at 3, 10, 30, or 50 mg/kg/dayand 2.5 mL/kg/dose (FIG. 16). Doses were administered for 9 days (first18 females/group) or 12 days (second 18 females/group) (FIG. 16). Bloodwas withdrawn at 9-day and 12-day time points. Concentrations werequantified by reverse-phase HPLC.

For each concentration, the samples were prepared by adding Labrasol tosolid R enantiomer or racemate in a beaker, sonicating to break upvisible aggregates, and magnetic stirring until a uniform appearance wasattained. The mixtures were transferred to a graduated cylinder andtopped off to the requisite volume using a Labrasol rinse of the beaker.The contents of the cylinder were mixed by inversion and stirredmagnetically until dosing.

These Labrasol dosing preparations utilized micronized R enantiomer, butnon-micronized racemate. To address the possibility that thenon-micronized racemate specimen might bias its dissolution rate inLabrasol, an independent experiment was performed. In this experiment,the solubility and appearance of 20 mg/mL preparations of micronized andnon-micronized racemate were examined. Micronized racemate (lotREH-28-9-1/PYAP-2-8-2-2M), non-micronized racemate (lot #PYAP-2-8-2-2),and micronized R enantiomer (lot #REH-28-4-2) were each suspended inLabrasol, swirled by hand, and then sonicated in a Branson 2100 tabletop sonicator at 25-28° C. for 3×5 min intervals and a 1×10 mininterval, interspersed with swirling by hand. The R enantiomer readilydissolved while both the micronized and non-micronized racemate mixturesremained cloudy with suspended fine particles (photos not shown). Byvisual inspection, the quantity of undissolved material in micronizedand non-micronized racemate samples was substantially the same.Microscopy (FIG. 17) revealed that most particles in both micronized andnon-micronized racemate suspensions were below 25 microns (FIG. 17).Some larger crystal fragments remained in the non-micronized sample.Since both micronized and non-micronized samples of racemate result insimilar amounts of undissolved material, it appears that the majordifference between R enantiomer and racemate Labrasol preparations isthe intrinsic solubility rather than the starting particle size of thesolute.

Mouse blood serum levels as a function of time were measured byquantitative HPLC. The R enantiomer in Labrasol attains significantlyhigher blood serum levels at the higher doses of 30 and 50 mg/kg/day (12and 20 mg/mL Labrasol administered). Racemate blood serum levels remainsubstantially lower (<4-5 fold AUC) (FIGS. 18 and 19). The initialparticle size differences between racemate an R enantiomer may accountfor a portion of the serum level differences. Also, the greaterintrinsic solubility of the R enantiomer over the racemate in Labrasolmay account for a portion of the serum level differences. Furthermore,serum level differences may be due to differences in membranepermeability (per Caco-2 cell and MDCK cell experiments; FIGS. 13 and14), absorption, distribution, or excretion.

Example 72 Comparison of the R Enantiomer and Racemate of2-Ethyl-3-methoxy-benzoic AcidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide for In VivoInduction of Transgene IL-12 Production from Gene-Modified Tumor Cells

The transgene tested in this example was murine IL-12 under the controlof the RTS. Mouse melanoma cells (B16) were transduced with anadenoviral vector Ad-RTS-mIL-12 that carries the murine IL-12 transgeneunder the control of RTS. These cells were injected subcutaneously(s.c.) into the C57BL/6 mice (histocompatible with B16 cells) and themice were treated with different doses of the R enantiomer and racemic2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide for threedays. The control groups included the mice receiving untransduced B16cells and the higher dose of ligand and another group receiving thetransduced B16 cells and no ligand. To assess the transgene expressiondirectly induced by the ligands, the tumors and serum were collectedfrom half of the mice from each group 72 hours after the subcutaneousinjection. Tumor extracts and serum were prepared and assayed for IL-12by ELISA.

The remaining mice were used for testing the transgene turn-off when theligand administration is discontinued for an additional 72 hours. Theresults indicate a difference in the transgene IL-12 expression in thetumor and the serum levels in the mice treated with the racemate and theR enantiomer of the ligand. The discontinuation of the ligandadministration resulted in the transgene turn off in all the groups in asimilar fashion.

Materials and Methods: B16 cells were infected with Ad.-RTS-mIL-12(MOI=100) in vitro. After 48 h, 5×10⁵ (B16, right flank) cells wereinjected s.c. into C57 BL/6 mice (10 mice/group). The R enantiomer andracemate ligands were dissolved in Labrasol by heating a 10 mg/mLmixture at 50° C. for 5 minutes. Both ligands were dissolved in Labrasolbased on visual inspection. Activating ligands were provided via oralgavage at the following doses (0 mg/kg; 3 mg/kg; 10 mg/kg; 30 mg/kg and50 mg/kg) daily for 3 days. Additional controls included B16-bearingmice receiving ligand only (50 mg/kg/day for 3 days and untreatedtumor-bearing mice). Five mice/group were harvested for isolation oftumor lesions and peripheral blood. Tissue was homogenized in 1 ml totalof phosphate-buffered saline. Lysate/serum was cryopreserved at −80° C.The alternate 5 mice/group were allowed to sit for an additional 72 h inthe absence of further ligand administration. These latter 5 mice/groupwere harvested for isolation of tumor lesions and peripheral blood.Tissue was homogenized as above, and cryopreserved and lysate/serum wascryopreserved at −80° C. Specimens were thawed at 37° C. in water bathand analyzed for mIL-12p70 and also mIFN-γ, a corollary IL-12-dependentcytokine important for CTL trafficking into tumors, using specificELISAs (BD-Pharmingen).

Results: The transgene IL-12 expression in the tumor as well as theserum levels followed a dose-dependent pattern for both the ligands(FIGS. 24 and 25). However, the expression levels were 2.5 to 3 foldhigher in the mice treated with the R enantiomer of2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide. Thispattern was observed in the tumor extracts and the serum. The controlgroups in which the mice received either the untransduced B16 cells plusthe maximum dose of the ligand, or transduced B16 cells without ligandadministration did not show any IL-12 expression. In the groups in whichthe ligand treatment was discontinued for 3 days, the expression levelsdropped to similar proportions in all the treatment groups, indicatingthat the ligand is cleared and the RTS activation is reversible when theligand is withdrawn. When the IFN-γ levels were assayed in the tumor andthe serum, the R-enantiomer group clearly showed enhanced expression.

Example 73 Pharmaceutical Composition Comprising2-Ethyl-3-methoxy-benzoic AcidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide

Table 1 shows a pharmaceutical composition comprising2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide, PL90G,vitamin E TPGS, BHT, and Miglyol 812N. PL90G is a highly purifiedphosphatidylcholine from soybean. The manufacturer of PL90G isPHOSPHOLIPID GmbH, a subsidiary of Lipoid. Vitamin E TPGS is a GRAS(Generally Recognized as Safe) compound and conforms to all requirementsof the current National Formulary (NF) monograph. Miglyol 812N containstriglycerides of the fractionated plant C₈ and C₁₀ fatty acids. Thefatty acids used for the production of Miglyol 812N are classified asGRAS and meet the requirements of the current NF and EP monographs forMedium-Chain Triglycerides. BHT [MeC₆H₂(CMe₃)₂OH] is an organic compoundthat is a lipophilic (fat-soluble) phenol which is widely used as anantioxidant food additive. The formulation was dispensed into hardgelatin capsules.

Example 74 Efficacy in Animal Models of Disease

Murine or human dendritic cells (DCs) transduced with adenoviral vectorencoding for RTS and mIL-12 or hIL-12, respectively, express cytokine invitro in the presence of (R)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide in a dosedependent fashion. In the absence of (R)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide, IL-12expression is at a basal level.

A comparative analysis of various administration routes and doses of(R)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide on theanti-tumor efficacy of intra-tumorally delivered DCs transduced with anadenoviral vector encoding murine interleukin-12 p70 (DC-SP1(IL-12p70))were carried out in a B16 melanoma mouse model. Results indicated thatanti-tumor efficacy was demonstrated with (DC-SP1(IL-12p70)) therapy incombination with (R)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide administeredby intraperitoneal (i.p.) injection, oral gavage or diet admix. Optimalanti-tumor effects occurred at ≧30 mg/kg (R)-2-ethyl-3-methoxy-benzoicacid N′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide doseadministered with (DC-SP1 (IL-12p70)) therapy.

In a murine B16 melanoma model in which mice were inoculated with tumorprior to the initiation of treatment (C57/BL6 mice were givensubcutaneous injections of B16 cells to form tumors), transduced AdDCscarrying murine IL-12 genes injected into the tumor mediated a completeregression of tumor and prolonged animal survival that was dependentupon systemic administration of (R)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide within 48hours following AdDCs injection. This drug dependent effect wasassociated with: transgene expression in the tumor and DLN; prolongedAd-DCs survival in the tumor microenvironment; migration and persistenceof AdDCs in the DLN; and induction of anti B16 CD8+ T cells.

In the above murine B16 melanoma model, intratumoral therapy consistingof an injection of 10⁷ AdDCs in combination with a 13 day treatment with(R)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide showedprotective immunity that was tumor-specific. When tumor-free animalswere re-challenged, on day 50 (post-initial tumor inoculation), theseanimals were refractory to B16 melanoma, but not MC38 colon carcinoma,progression.

Example 75 Replication Incompetent Recombinant Adenovirus Harboring RTSand hIL-12

FIG. 23 is a diagrammatic representation of Ad-RTS-hIL-12 in which theE1 and E3 regions have been deleted and the RTS-IL12 components replacethe E1 region (FIG. 23 is not drawn to scale). The terms used in FIG. 23have the following meanings:

RTS-IL-12: Human IL-12 under the control of the RheoSwitch® TherapeuticSystem

P_(reg): Promoter driving the IL12 expression, regulated by theactivated drug

hIL-12: Coding sequences for the p40 and p35 subunits of human IL-12separated by an IRES sequence

polyA: Polyadenylation signal

P_(UbC): Ubiquitin C promoter

VP16-RXR: Fusion between VP16 transcriptional activation domain and achimeric RXR

IRES: Internal Ribosome Entry Site

Gal4-EcR: Fusion protein between Gal4 DNA binding domain and ecdysonereceptor ligand binding domain

Ad: Adenovirus-5 backbone containing E1 and E3 gene deletions.

The recombinant adenoviral vector Ad-RTS-hIL-12 is prepared as describedby Anderson et al., Gene Therapy, 4: 1034-1038 (2000). The Ad-RTS-hIL-12vector is produced in the following manner. The coding sequences for thereceptor fusion proteins, VP16-RXR and Gal4-EcR separated by theEMCV-IRES (internal ribosome entry site), are inserted into theadenoviral shuttle vector under the control of the human ubiquitin Cpromoter (constitutive promoter). Subsequently, the coding sequences forthe p40 and p35 subunits of hIL-12 separated by IRES, placed under thecontrol of a synthetic inducible promoter, are inserted upstream of theubiquitin C promoter and the receptor sequences. The shuttle vectorcontains the adenovirus serotype 5 sequences from the left end to mu16,from which the E1 sequences are deleted and replaced by the RTS andIL-12 sequences (RTS-hIL-12). The shuttle vector carrying the RTS-hIL-12is tested by transient transfection in HT-1080 cells for liganddependent IL-12 expression. The shuttle vector is then recombined withthe E3-deleted adenoviral backbone by cotransfection into HEK 293 cellsto obtain recombinant adenovirus Ad-RTS-hIL-12. The clinical gradeadenoviral vector is produced in a cGMP facility.

Example 76 Transduction of Autologous Immature Dendritic Cells byAdenovirus Containing hIL-12 Transgene and Rheoswitch® TherapeuticSystem

Harvesting of PBMC by leukapheresis: Subjects undergo a 90-120 minuteleukapheresis at the Hillman outpatient CTRC. The leukapheresisprocedure involves the removal of blood from a vein in one arm; thepassage of blood through a centrifuge (cell separator), where itscomponents are separated and one or more components are removed; and thereturn of the remaining components to the subject's vein in the same orother arm. No more than 15% of the subject's total blood volume iswithdrawn at any one time as blood is processed through the cellseparator device. In the cell separator, blood is separated into plasma,platelets, white cells and red blood cells. White blood cells (WBC) areremoved and all the other components are returned into the subject'scirculation. Every attempt is made to use two peripheral IV lines forthis procedure. If that is not possible, a central line may benecessary. The subject has to be cleared by physician to undergoleukapheresis, and is routinely screened for vital signs (includingblood pressure) prior to the procedure.

Processing: After collection, the leukapack is delivered by hand to theCPL, and is immediately processed by centrifugal elutriation in ELUTRA™.This is a closed system validated for clinical use. The monocytefraction is recovered, and after the recovery and viability of cells areestablished, they are transferred to an Aastrom cartridge for 6-dayculture in the presence of IL-4 and GM-CSF. All processing and washingprocedures are performed under sterile conditions.

Initial plating: Monocytes recovered from a single leukapack are countedin the presence of a trypan blue dye to determine the number of viablecells. Monocytes are evaluated for purity by flow cytometry. Monocytesare resuspended in serum-free and antibiotic-free CellGenix medium,containing 1,000 IU/mL of IL-4 and 1,000 IU/mL of GM-CSF perSOP-CPL-0166, and placed in an Aastrom cartridge. A minimum loadingvolume of 50 ml and a minimum cell number are required for cassetteinoculation.

Culture: The Aastrom cartridge is placed in the incubator in theReplicell System, a fully closed, cGMP-compatible automated culturedevice for immature DC generation.

Immature DC harvest: On day 6, the Aastrom cartridge is removed from theincubator and immature DCs are harvested. The cells are recovered bycentrifugation at 1,500 rpm, washed in CellGenix medium, counted in thepresence of a trypan blue dye and checked for morphologic and phenotypiccharacteristics.

Viability: This is determined by performing hemocytometer cell counts inthe presence of trypan blue. Generally, >95% of harvested cells areviable, i.e., exclude a trypan blue dye. If viability is less than 70%the immature DCs will be discarded.

Phenotyping: The cells generated in culture are counted by microscopicobservation on a hemocytometer, and a preliminary differential count (DCvs. lymphocytes) is obtained using a trypan blue dye. Confirmation ofthe differential count is made by flow cytometry, gating on DC vs.lymphocytes and using high forward and side scatter properties ofimmature DC as the criterion for their identification. Immature DCsroutinely contain >80% of cells with dendritic cell morphology and haveDC phenotype.

IL-12p70 potency assay: It has been established that mature DCs (mDCs)have the ability to produce IL-12p70 spontaneously or upon activationwith CD40L with or without addition of innate immunity signals (e.g.,LPS). A standardized IL-12p70 production assay was recently establishedand is applicable to small samples or large lots of DC vaccinesgenerated under a variety of conditions. The current potency assayconsists of two distinct steps, the first involving co-incubation ofresponder DCs with J588 lymphoma cells stably transfected with the humanCD40 ligand gene as stimulators. The second step involves testing ofsupernatants from these co-cultures for levels of IL-12p70 secreted byDCs stimulated with J558/CD40L+/−LPS in the Luminex system. This potencyassay has an inter-assay CV of 18.5% (n=30) and a broad dynamic range,which facilitates evaluation of various DC products characterized byvastly different levels of IL-12p70 production. The normal range for theassay established using DC products generated from monocytes of 13normal donors was 8-999 pg/mL, with a mean of 270 pg/mL

Example 77 Adenovirus Transduction of Immature DCs

Immature DCs are harvested, counted and tested for viability.Approximately 6-7×10⁷ DCs are transduced with the adenoviral vector atthe optimal multiplicity of infection (the optimal MOI, between 500 and1000, to be finalized) for the optimal viral adsorption time (between 2h and 4 h, to be finalized). After transduction, the cells are washedrepeatedly to remove any unadsorbed viral particles. Aliquots are setaside for sterility, endotoxin, mycoplasma, DC Phenotype and IL-12transgene induction assays. The target dose of 5×10⁷ cells is held at 4°C. in saline until release testing is complete.

The transduced DCs (AdDCs) which have passed all release testing aredelivered to the clinic for patient administration as described below.

The in vitro testing of the IL-12 transgene induction by the transducedAdDCs takes a minimum of two days for the results to be available andtherefore IL-12 production in response to (R)-2-ethyl-3-methoxy-benzoicacid N′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide willnot be a release criterion for the intratumoral injection of the AdDCs.However, based on preliminary studies with AdDCs derived from threehealthy volunteers, the induced IL-12 expression in response to(R)-2-ethyl-3-methoxy-benzoic acidN′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide has beenachieved within a target range of 80 to 300 ng IL-12 per million cellsin 24 hours (expression level of 80 ng/million cells/24 h was observedin 2 out of the 3 AdDC preparations). Therefore, the in vitro IL-12induction results are used for analysis of the treatment outcome. Assuch, the transduced AdDCs are used for injection after viraltransduction and washing. Any left-over untransduced and transduced DCsare cryopreserved for future analysis as necessary.

Administration of the AdDCs to patients: A single intratumoral injectionof AdDCs in 0.9% saline solution that contains approximately 5×10⁷ cellsper injection (or approximately 90% of the DC yield from the trialsubject) in a volume of 150 microliters. Subjects should be dosed withthe maximum feasible dose of AdDCs, not to exceed 5×10⁷ cells perinjection.

Example 78 Administration to Humans of Adenovirally TransducedAutologous Dendritic Cells Engineered to Express hIL-12 Under Control ofThe RheoSwitch® Therapeutic System in Subjects with Stage III and IVMelanoma

Cohort 1 will consist of 12 subjects and cohort 2 will consist of 28subjects all who will receive a single intratumoral injection oftransduced autologous AdDCs approximately 24 hours after the first doseof (R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide. Cohort1 subjects will be placed into 4 groups of 3 subjects each (A, B, C, D)to receive escalating doses of (R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide. Cohort2, subjects will receive the maximum tolerated oral dose of(R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide (asdetermined from cohort 1). Immediately following the injection of AdDCs,subjects will be treated with thirteen additional daily doses of(R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide. Safety,tolerance, and transgene function will be assessed for all subjects ineach group of cohort 1 up to one month after injection of AdDCs beforeenrolling subjects to receive the next higher dose of(R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide. Thirtydays after cohort 1 has completed the inpatient treatment phase and atleast six to eight weeks following the intratumoral injection oftransduced autologous dendritic cells, if the subject desiresretreatment and the subject meets the criteria for a retreatment,additional injection(s) of DCs with 14 consecutive daily doses of(R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide may beadministered. The dose of (R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide (0.01mg/kg, 0.1 mg/kg, 1.0 mg/kg or 10.0 mg/kg) will be the maximum tolerateddose from cohort 1.

Patients will be assessed by physical examinations (including ECOGperformance status), vital signs, serum chemistry, urinalysis,hematology, adverse events, antibodies and cellular immune response toadenoviral vector and components of the RheoSwitch® Therapeutic System.Also assessed will be single dose and steady-state pharmacokinetics/ADMEof oral (R)-3,5-dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide and itsmajor metabolites, analysis of transgene function through measurement ofhIL-12 levels in biopsies of the target tumor and/or draining lymphnodes, evaluation of the immunological effects by measurement of thecellular immune response (T cells) in biopsies of target tumor, draininglymph nodes and peripheral circulation, and a serum cytokine profile.

It is to be understood that the foregoing described embodiments andexemplifications are not intended to be limiting in any respect to thescope of the invention, and that the claims presented herein areintended to encompass all embodiments and exemplifications whether ornot explicitly presented herein.

1. A compound selected from the group consisting of:(R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;(R)-3,5-Dimethyl-benzoic acidN′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide; (R)-3,5-Dimethyl-benzoicacid N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide;(R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide;(R)-3,5-Dimethyl-benzoic acidN′-(2-bromo-benzoyl)-N-(1-tert-butyl-butyl)-hydrazide;(R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide;(R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide;(R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-chloro-pyridine-3-carbonyl)-hydrazide;(R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(6-chloro-pyridine-3-carbonyl)-hydrazide; and(R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide. 2.(R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide havingan enantiomeric excess of at least 95% or a pharmaceutically acceptablesalt thereof.
 3. (R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide havingan enantiomeric excess of at least 99% or a pharmaceutically acceptablesalt thereof.
 4. A pharmaceutical composition comprising the compound ofclaim 1 and a pharmaceutically acceptable carrier.
 5. A pharmaceuticalcomposition comprising the compound of claim 2 or 3 and apharmaceutically acceptable carrier.